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 Published by
World Scientific Publishing Europe Ltd.
57 Shelton Street, Covent Garden, London WC2H 9HE
Head office: 5 Toh Tuck Link, Singapore 596224
USA office: 27 Warren Street, Suite 401-402, Hackensack, NJ 07601

Library of Congress Cataloging-in-Publication Data

Names: Arrabito, Giuseppe, editor. | Wang, Liqian, editor.
Title: DNA nanotechnology for bioanalysis : from hybrid DNA nanostructures to
functional devices / edited by Giuseppe Arrabito (University of Palermo, Italy),
Liqian Wang (Chinese Academy of Sciences, China).
Description: New Jersey : World Scientific, [2017] | Includes bibliographical references.
Identifiers: LCCN 2017013235 | ISBN 9781786343796 (hc : alk. paper)
Subjects: LCSH: DNA--Biotechnology. | Nanobiotechnology.
Classification: LCC QP624 .D165 2017 | DDC 572.8/6--dc23
LC record available at https://lccn.loc.gov/2017013235

British Library Cataloguing-in-Publication Data

A catalogue record for this book is available from the British Library.

Copyright © 2018 by World Scientific Publishing Europe Ltd.

All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means,
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Dedication

Giuseppe Arrabito would like to dedicate this book to all the colleagues of
the research group of Prof. Bruno Pignataro for their support during the
writing phase. Giuseppe Arrabito would like to thank to Prof. Weihua Han
(Lanzou University, China) for useful discussion.
Giuseppe Arrabito would finally like to dedicate this book to his
­parents, for their unconditional love.

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 9”x6” b2875  DNA Nanotechnology for Bioanalysis: From Hybrid DNA Nanostructures to Functional Devices

Foreword

Deoxyribose Nucleic Acid (DNA) is the molecule that contains the life

code as a “blue print” in all living systems. DNA is a perfect material for
building nanostructures because of the special Watson–Crick pairing rule
involving four fundamental bases (A, T, G, C). Each base can be consid-
ered as a basic brick for designing nanostructures. The concept is very
much like a LEGO methodology of constructing a structure from basic
bricks. Even if the basic elements are restricted to four, we can program
an infinite number of different sequences of such elements and therefore,
using the pairing rule, create an infinite number of structures.
It has been over 30 years since the pioneering work by Professor
Nadrian Seeman of New York University in the 1980s. We have seen a
tremendous change in this field, DNA nanotechnology is not just beautiful
nanoscaled structures, it holds great promise in life science applications.
This book has been written by the researchers in different groups in
Europe and China who work in the field. The presentation is lucid and sim-
ple; it is easily understandable by beginners and undergraduate students. This
book provides both a solid background introduction and most advanced
applications. I trust that students and researchers who are not in the field will
find this book a useful introduction to DNA nanotechnology for bioanalysis.

Prof. Chunhai Fan

Laboratory of Physical Biology, Shanghai Institute of Applied Physics,
Chinese Academy of Science, Shanghai 201800, China

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Preface

Writing a book about the latest advances in DNA nanotechnology is

­definitely a very difficult task, since this is one of the most rapidly grow-
ing scientific topics and it is destined to produce a tremendous amount of
new fundamental concepts in the world of Nanoscience and new class of
molecular devices allowing for advances in Chemistry, Biology and
Medicine. One has to consider that 542 papers about G quadruplex and
1564 papers (272 according to web of science) about DNA origami have
been published in 2016, according to Scopus database. This means that
the idea to provide a comprehensive view of the field is challenging.
The idea that originated the book came up whilst the editors,
G. Arrabito and L. Wang, published a Critical Review on The Analyst
which, for the first time, reported on applications of DNA nanotechnology
in the bioanalytical sciences. Following up that work, they had the oppor-
tunity to cover this exciting field under another perspective — with the
hope to reach a different readership, especially students and researchers
who are not specifically expert in the DNA nanotechnology and its
applications.
We here tried to explore the general aspects of functional DNA nano-
structures such as DNA tiles, DNA origami, DNA quadruplexes, DNA-
based bio- and inorganic covalent immobilization for biosensing
applications and analytical sciences. We included the state of the art of
important methods used for the mild, biocompatible and programmable

ix

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x Preface

fabrication of several different types of DNA nanostructures and hybrid

nanostructures bearing proteins, molecules or inorganic moieties cova-
lently binded onto DNA via self-assemblage processes. All the fabricated
structures here reported are discussed according to the attributes that are
of specific interest for applications in fields like biotechnology, biosens-
ing, electrochemistry, molecular mimicking, diagnostic, biomedicine and
analytical biochemistry.
The book can be considered as a clear and concise introduction to the
field and is specifically focused on the new applications of DNA nano-
technology in sensors and Chemistry, especially in the field of Analytical
Chemistry in which scientists are just starting to think about usability of
DNA nanostructures. In fact, most of the papers published so far are in the
material science field (750), whereas a fewer amount (30) is in the
Analytical Chemistry category.
A part from providing an introduction into the exciting field of DNA
nanosciences, the main aim of the book is the one to define some points
of future development in the field, mainly — we concluded — driven by
the need to make such technology more useful and cost-effective by solv-
ing issues like the high cost of oligos synthesis, better means of detection
and efficient integration into output devices in order to fully show its
potentiality in real world applications. We believe that DNA nanotechnol-
ogy is still not at his mature age and more breakthroughs still have to
come in the future. We wrote this book with the aim to encourage students
and scientists to bring their efforts into this field. The book has been
intended as written for the generalist at a level that could be understood
by an undergraduate student in Physics, Chemistry or Biology or by sci-
entists who are not in this field. We try to provide “first principles” intro-
duction to each of the proposed approaches, exploring its attributes and
hurdles along with clear working examples and real applications in a labo-
ratory of chemistry. A part from giving a comprehensive view of the field,
we try to indicate possible future developments, especially by considering
the electrochemical and optical mediated detection methods that can make
this technology amenable for integration into future analytical devices.
The Editors are sincerely thankful to all the authors of the chapters,
without their passion for their work, this book would not have been pos-
sible. The Editors wish to formulate sincere thanks to Prof. Bruno

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Preface xi

Pignataro, Full Professor in Physical Chemistry at the University of

Palermo for having transferred the sincere passion for nanosciences and
Physical Chemistry.
Dr. Arrabito acknowledges Prof. Christof Niemeyer, Professor of
Chemical Biology at Karlsruhe Institute of Technology (KIT) and Director
of the Institute for Biological Interfaces (IBG-1), for having permitted
Dr. Arrabito to get interest DNA nanostructures and its applications in
Biology. Dr. Arrabito wishes to send special thanks to Dr. Christian
Falconi (Assistant Professor, University of Rome Tor Vergata) for gener-
ous support and possibility to work in the field of electronic biosensors.
Dr. Arrabito also wants to thank Prof. Francesco Ricci (Associate
Professor, University of Rome Tor Vergata) for the encouragements and
the ideas which motivated Dr. Arrabito to consider DNA aptamers as
potential breakthrough in biosensors field.
Dr. Wang would like to acknowledge Prof. Chunhai Fan for providing
her consistent direction, motivation and the opportunity to work in one of
the most advanced groups that deals with DNA nanotechnology. Dr. Wang
also wants to thank Dr. Piero Morales, who has introduced her into DNA
nanotechnology and teaching her through all her PhD years.
Without efforts and encouragements of the above cited professors and
the invaluable contribution of all the authors of the book, nothing of this
would have not been possible. And maybe new generation of scientists
would not have got any idea of how great DNA nanoscience can be!

Giuseppe Arrabito
Department of Physics and Chemistry,
University of Palermo, Viale delle Scienze,
Parco d’Orleans II, 90128 Palermo, Italy

Liqian Wang
Laboratory of Physical Biology,
Shanghai Institute of Applied Physics,
Chinese Academy of Sciences,
Shanghai 201800, China

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About the Authors

Giuseppe Arrabito received his PhD (2012) from Scuola Superiore di

Catania. He carried out Post-Doctoral fellowships in the group of C.M.
Niemeyer (2013) and in the group of C. Falconi (2014). He is currently a
Post-Doctoral Scientist in the group of B. Pignataro. His main research
interests are in the fields of bioanalytical chemistry, micro- and nano-
biological array fabrication, Dip-Pen Nanolithography, DNA-directed
material assembly for nanotechnological applications and ZnO nanostruc-
tures for biosensing.

Liqian Wang received her PhD degree (2016) in Materials Science from
the University of Rome Tor Vergata. She worked as a visiting researcher
(2014) in the group of Ilia N. Ivanov at Oak Ridge National Laboratory.
She is currently a Post-Doctoral Scientist in the group of Chunhai Fan.
Her research interests include the application of DNA nanostructures as
nanoelectronic devices and the interface between organic self-assembly
and inorganic nanotechnology.

xiii

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Contents

Forewordvii
Prefaceix
About the Authorsxiii

Chapter 1 Nanotechnology and the Unique Role of DNA 1

 Elisa-C. Schöneweiß, Andreas Jaekel
and Barbara Saccà
1.1 Introduction 1
1.2  The DNA Molecule 4
1.2.1  The double helical B-form 4
1.2.2  Alternative DNA forms 6
1.2.3  Physical and chemical properties of the DNA 8
1.3 DNA as Building Block of Self-assembled Nanostructures 8
1.4  Functionalization of DNA Nanostructures 11
1.4.1  DNA as a recognition motif 12
1.4.2  Synthesis of DNA–protein conjugates 14
1.4.2.1  Covalent coupling 14
1.4.2.2  Non-covalent coupling 16
1.5  Dynamic DNA Motifs 16
References19

xv

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xvi Contents

Chapter 2 DNA as Building Material at the Nanoscale:

From Concepts to Software-aided Design 27
Wolfgang Pfeifer, Georg Homa, Giuseppe Arrabito
and Barbara Saccà
2.1  Basic Concepts of Design 28
2.2  The Two “Schools” of DNA Design 29
2.3  The Multi-stranded Approach 33
2.3.1  The first immobile junction designed by SEQUIN 33
2.3.2  Tile-to-tile assembly 38
2.3.3  Platonic and Archimedean tilings 41
2.3.4  Single-stranded tiles 41
2.4  Scaffold-based Designs 42
2.4.1  Monolayer DNA origami 42
2.4.2  Space-filled DNA origami 44
2.4.3  Wireframe DNA origami 46
2.5  DNA Aptamers: From Basics to Applications 49
References51

Chapter 3 DNA Sensors for the Detection of Biomolecules

and Biochemical Conditions 57
Birgitta R. Knudsen, Anni H. Andersen,
Magnus Stougaard, Giuseppe Arrabito,
Raffaella Suriano and Yi-Ping Ho
3.1 Introduction 58
3.2  Construction of DNA Sensors 59
3.2.1  Top-down fabrication of DNA sensors 60
3.3  Signal Transduction Mechanisms 64
3.3.1  Fluorescence-based sensing 64
3.3.1.1  Förster resonance energy transfer 65
3.3.2  Plasmonics-based sensing 66
3.3.3  DNA nanopore-based sensing 69
3.4  Detection of Nucleic Acids 71
3.5  Detection of Proteins 73
3.6  Detection of Enzymes 74
3.7  Detection of Biochemical Conditions and Small Molecules 79

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Contents xvii

3.8  Conclusion and Future Perspective 85

Acknowledgements85
References85

Chapter 4 DNA Nanostructures in Cell Biology and Medicine 99

Liqian Wang and Giuseppe Arrabito
4.1 Introduction 100
4.2  Cell-SELEX for Cellular Detection 100
4.3 Single Cell Manipulation by DNA-directed
Protein Immobilization 104
4.3.1  DNA-directed immobilization 104
4.3.2 Dip-pen nanolithography coupled with DDI
for single cells interaction arrays 106
4.3.3 High-throughput patterning methodologies
for single cells interaction arrays 110
4.4  How to Get into Cells 112
4.4.1 Phagocytosis 112
4.4.2 Pinocytosis 114
4.4.3 Macro-pinocytosis 114
4.4.4  Clathrin-mediated endocytosis 114
4.4.5  Caveolae-mediated endocytosis 115
4.4.6  Clathrin- and caveolin-independent endocytosis 116
4.5  DNA Nanostructures in Cellular Environment 116
4.5.1  DNA structures in cell lysates 117
4.5.2  DNA structures in serum 117
4.6  DNA Carriers for Drug Delivery 118
4.7  Conclusion and Perspectives 121
References122

Chapter 5 Targeting G-quadruplex DNA as Potential

Anti-cancer Therapy 129
Riccardo Bonsignore, Elisa Trippodo and
Giampaolo Barone
5.1 Introduction 129
5.2  Structural Details 130

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xviii Contents

5.3  Biological Involvement 133

5.3.1  G-quadruplexes in human oncogene promoters 133
5.3.2  G-quadruplexes in telomeres 134
5.4  Recent Advances in Targeting G-quadruplex DNA 135
5.4.1  Targeting G-quadruplex DNA with organic binders 137
5.4.2  Targeting G-quadruplex DNA with metal complexes 147
5.5  Conclusion and Perspectives 155
References156

Chapter 6 DNA-aided Super-resolution Bioimaging 163

JiaJun Li, Giuseppe Arrabito and ZhaoShuai Gao
6.1 Introduction 163
6.2  Diffraction Limit 165
6.3  Stimulated Emission Depleting Microscope 166
6.4  Localization Microscopes 170
6.5  Structured Illumination Microscope 173
6.6  Structured Overview about Super Resolution Technologies 175
6.7  DNA-nanostructures Aided Super Resolution Imaging 176
6.7.1  Binding-activated localization microscopy 176
6.7.2 Binding-DNA-aided point accumulation
for imaging in nanoscale topography 178
6.7.2.1 Points accumulation for imaging
in nanoscale topography 178
6.7.2.2  DNA-aided PAINT 180
6.8 Conclusion 185
References185

Conclusion189

Index193

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Chapter 1
Nanotechnology and the Unique
Role of DNA

Elisa-C. Schöneweiß, Andreas Jaekel and Barbara Saccà*

Centre for Medical Biotechnology (ZMB)

University of Duisburg-Essen
45117 Essen, Germany
*barbara.sacca@uni-due.de

This chapter is a clear introduction to the role of DNA molecule in

Nanotechnology. A part from describing the canonical and alternative
structures, this chapter is aimed at giving fundamentals of self-assembling
strategy of DNA nanostructures.

1.1 Introduction
“What I want to talk about is the problem of manipulating and controlling
things on a small scale.” With these words Richard Feynman introduced
his talk at Caltech on December 29th 1959, entitled “There is plenty of
Room at the Bottom” [1], which signed the beginning of a new field of
science: nanotechnology. In his visionary speech, the Nobel Laureate in

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2 E.-C. Schöneweiß, A. Jaekel and B. Saccà

Physics anticipated the tremendous possibilities offered by the

­“staggeringly small world that is below,” some of which became reality
only recently. For the first time, scientists were challenged to overstep the
minimal size of things that can be manipulated, bringing miniaturization
to the smallest possible level. He proposed for example to use light to
arrange molecules or even single atoms in a desired pattern on a suitable
surface. This idea found later application in a series of top-down
approaches, such as photolithography, inject-printing and other micropat-
terning methods [2–4], all sharing the same principle: the exploitation of
the laws of physics to pack a huge amount of information on an exceed-
ingly small space. On the other hand, the opposite process, that is, the
extraction of all possible information from a tiny object, was also envi-
sioned as a realistic goal, achievable for example with the development of
more powerful microscopes. Nowadays, complex molecular assemblies
can be observed almost at atomic resolution thanks to the improvement of
electron microscopy [5–7] and kinetic processes can be resolved with
picosecond accuracy using advanced single-molecule techniques [8–11].
Storing and using the information carried in a tiny space is the way
nature performs its tasks. Fundamental biological processes such as cel-
lular duplication, growth, movement and interactions are all tightly regu-
lated in a microscopic and crowded area. What is mostly striking about it
is that the information driving the whole machinery is contained in a few
micrometer-large fraction of the cell, the nucleus, in the form of an almost
two meter-long chain, the DNA molecule, which carries about one bit of
information every 50 atoms. In this sense, the DNA molecule is probably
the most brilliant example of nanotechnology in nature, being itself a
nanosized object, capable to store and transmit the information necessary
to life in a simple and efficient way. It is therefore not surprising that the
beginning of the nanotechnology era started only few years after the dis-
covery of the structure and function of DNA by Watson and Crick in 1953
[12, 13] and that latest advancements in the field have been made using
DNA as the material.
The idea of employing DNA to construct molecular objects was pio-
neered by Ned Seeman in the early 1980s [14] and since then structural
DNA nanotechnology has rapidly evolved, both in terms of design princi-
ples and applications [15]. Using a simple four bases code and applying

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Nanotechnology and the Unique Role of DNA 3

predictable base pairing rules, this bottom-up approach allows the

­realization of DNA objects of almost any desired shape and pattern, thus
enabling to reach the nanosized world from the molecular level, with sub-
nanometer resolution. A notable breakthrough in the field occurred in
2006, when Paul Rothemund published the first scaffolded DNA-origami
approach [16]. This method revolutionized our vision of DNA as a con-
struction material, giving accessibility to nanosized objects with a level of
sophistication previously only hardly imaginable [17, 18]. Recently other
design strategies have emerged [19–22], enlarging the spectrum of attain-
able structures and demonstrating that the future of DNA design is still
bright. This, together with the availability of user-friendly design tools
and synthetic accessibility of oligonucleotides at relatively low costs,
­enabled the exponential growth of DNA nanotechnology in the past few
years [23].
Besides solving design challenges, scientists rapidly succeeded in
demonstrating the use of those structures for realistic applications, from
the development of addressable molecular pegboards for protein pattern-
ing [24–27] or encapsulation [28–32], to optoelectronic hybrid materials
[33] and organic catalysts [34]. Another field in great expansion is cou-
pled to the advancement of single-molecule technologies, enabling for
example the precise localization and counting of molecules in spatially
distributed samples or the disclosure of anomalous kinetic events occur-
ring on a time scale normally not accessible by standard methods [8,
35–40]. Recently, DNA nanostructures have also shown interesting prop-
erties in cells [41] and are currently explored as promising targeting and
delivery systems [42, 43]. For all these applications, the nucleic acid
structure must be previously functionalized, i.e. chemically modified with
small molecules or proteins [44]. At this purpose, not only classical DNA-
conjugation strategies have been implemented and revisited [45], but other
methods based on sequence-specificity recognition [46] and protein adap-
tors [47, 48] have been also successfully applied.
In addition to the use of DNA as a static framework for controlling the
spatial arrangement of molecules, the programmability of nucleic acids
structures can be conveniently employed to develop dynamic systems,
whose interconversion between distinct species occurs in a predictable
manner [49–51]. This principle has been successfully employed to

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4 E.-C. Schöneweiß, A. Jaekel and B. Saccà

develop bioinspired DNA-nanomotors [52, 53] and bioinformatic tools for

emulating complex reaction circuits [54].
Finally, one should not forget the enormous contribution to the field
of DNA nanotechnology given by Chad Mirkin, who firstly employed the
DNA molecule as a recognition motif attached over the surface of metal
nanoparticles for biosensing applications [55, 56]. Later, the same princi-
ple has been applied for linking distinct nanoparticles together, leading to
the generation of colloidal nanocrystals [57, 58] and nanoplasmonic mate-
rials with advanced optical properties [59].
In conclusion, although many of the initial Feynman’s predictions
became true, one cannot but remaining astonished by the enormous pro-
gresses made in the field and the rapid advancement of technologies and
design strategies to approach the nanosized world with such a high level
of control. In this sense, the DNA molecule represents an ideal candidate
for the realization of those self-organizational systems initially envisaged
in Feynman’s lecture, which can help us to manipulate matter and under-
stand the basic principles of self-assembly in natural and man-made
architectures.

1.2  The DNA Molecule

1.2.1  The double helical B-form
The fundamental building block of every DNA nanostructure is the DNA
double helix. Understanding the structure and chemical–physical proper-
ties of the double helix is therefore necessary when the purpose is to
design ordered assemblies of single DNA units and using them for further
functionalization.
DNA is a polymer composed by the periodic repetition of four pos-
sible nucleotides (Figure 1.1). Each nucleotide is made up of three com-
ponents: a phosphate group, a five-carbon sugar deoxyribose and a
nitrogenous base (also termed nucleobase or simply base). The four nucle-
obases are adenine (A), guanine (G), thymine (T) and cytosine (C).
Whereas the sugar–phosphate moieties provide the backbone of the
nucleic acid structure, the four bases are responsible for its helical shape.

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Nanotechnology and the Unique Role of DNA 5

Figure 1.1.    The DNA molecule in its most common B-form is a right-handed helical
structure, which is 2 nm wide and rises up ca. 3.4 nm every helical turn (corresponding to
ca. 10.5 base pairs (bp)). The four nucleobases (A, G, T, C) and their paring rule are also
indicated. Note that whereas AT pairs are stabilized by two hydrogen bonds, GC pairs
involve three bonds, which explains their higher contribution to helical stabilization.
Figure modified from open sources (PDB: 1DUF).

This latter comes mostly from base stacking interactions among the aro-
matic nitrogenous bases. This is true for both single-stranded and double-
stranded DNA. The double-stranded (ds) form, however, requires hydrogen
bonding between the bases of each strand in order to form a stable double
helix. The process is called base pairing because each nucleobase pairs
only with a certain other nucleobase, named “complement”: A pairs with
T and G pairs with C. Such a Watson–Crick base-pairing rule is the fun-
damental law of each DNA design: once given a certain DNA sequence,
its complementary sequence is uniquely defined. Such a reliable

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6 E.-C. Schöneweiß, A. Jaekel and B. Saccà

programmability of DNA nanostructures basically represents the main

reason for the rapid and successful implementation of structural DNA
nanotechnology.
In its B-form, the DNA is a right-handed double helix, about
2 nm wide and with a helical pitch of 3.4 nm comprising ca. 10.5 bp. The
structural features of the glycosidic bonds of two pairing strands cause
them to twist around the helical axis forming two unequally spaced
grooves, a major groove (2.2 nm wide) and a minor groove (1.2 nm wide).
Although B-DNA is the most predominant form in cells, at least other
two helical conformations are possible, A-DNA and Z-DNA, with
strongly different geometry and dimensions. Whereas A-DNA mainly
occurs in non-physiological conditions and adopts a wider (2.3 nm) and
shorter helical pitch (ca. 2.8 nm/turn), the Z-form may appear upon DNA
methylation and is a left-handed helix with a narrow diameter (1.8 nm)
and a high helical pitch (4.6 nm/turn). Because of its abundance and sta-
bility, the B-DNA is the most widely form employed for construction of
DNA nanostructures.

1.2.2  Alternative DNA forms

Non-canonical DNA motifs of particular importance in DNA nanotech-
nology are also hairpin loops, G-quadruplexes, i-motifs and triplex DNA.
Hairpin structures are formed by intra-strand base pairing, occurring
when the sequence contains two inverted repeats, also termed palindromes
(Figure 1.2(a)) [60]. The stability of this motif depends on the length of
the double-helical segment (also termed stem), its sequence, as well as
from the loop. This is the single-stranded region of the motif, which
allows the sequence to fold back on itself to form the stem. Optimal loop
lengths are about 4 to 8 bases long. Hybridization of the hairpin motif with
a complementary sequence, which is also forming a hairpin, leads to a
longer and therefore more stable double-helical segment. This conforma-
tional change can be easily programmed and in certain conditions even
reversed, thus making hairpin motifs attractive structural features for
dynamic DNA devices. Recently, the organized tethering of a distinct
number of hairpin loops at precise locations of a DNA nanostructure has
been advantageously used for actuation of large-scale translations [53].

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Nanotechnology and the Unique Role of DNA 7

Figure 1.2.    Alternative DNA motifs, largely employed in DNA nanotechnology. (a) The
hairpin motif, formed by a duplex stem connected at one end by a single-stranded loop,
(b) a G-quadruplex structure, stabilized by Hoogsteen hydrogen bonds between guanine
bases and (c) a triple helix, formed by an oligopyrimidine single-stranded DNA binding to
the major groove of an oligopurine sequence of the target duplex DNA. Figures adapted
by open sources (PDB: 1JVE, 1C35 and 1BWG, respectively).

Widely employed in DNA nanotechnology are also G-quadruplexes

(G4) and i-motifs. G4 structures are based on the stacking of several so-
called G-quartets, which consist each of four guanine bases held together
by Hoogsteen-type hydrogen bonding (Figure 1.2(b)) [61]. G4 structures
are further stabilized by the presence of cations (generally sodium and
potassium) in the central channel of the helix. A G4 motif can be formed
by one or several DNA strands, oriented either in a parallel or antiparallel
fashion. The structural diversity, folding properties and stabilities of
G-quadruplex DNA have been extensively studied, both as a model for a
non-canonical secondary DNA structure and as a pharmacological target
for small molecules that have potential to impact gene expression [61].
i-Motifs are four-stranded DNA secondary structures which can form
in cytosine rich sequences [62]. Stabilized by acidic conditions, they are
made of two DNA duplexes held together in an antiparallel orientation by

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intercalated, cytosine–cytosine+ bps. By virtue of their pH dependent

­folding (in slightly acidic conditions), i-motif forming DNA sequences
have been used extensively as pH switches for applications in nanotech-
nology [63]. 
Finally, another non-conventional DNA structure is the triple helix,
constituted by a single DNA strand, normally termed triplex-forming oli-
gonucleotide (TFO), wrapped around a B-form double helix through
Hoogsteen hydrogen bonds (Figure 1.2(c)). A TFO binds to the major
groove of the target duplex DNA in a sequence-specific manner, generat-
ing base triplets with the purine bases. Therefore, formation of a triple-
helix is limited by the presence of oligopyrimidine • oligopurine sequences
in the DNA target. TFOs have been recently the focus of considerable
interest in nanotechnology [64] for developing new molecular biology
tools as well as diagnostic agents [65].

1.2.3  Physical and chemical properties of the DNA

Besides its predictable self-recognition properties, at least other two rea-
sons have made the DNA molecule a supreme candidate as a construction
material: (i) its flexibility (the double helix has a persistence length of
50 nm) and (ii) its easy synthetic accessibility and modification. The for-
mer enables to intertwine distinct DNA chains together into many differ-
ent forms, thus enormously enlarging the spectrum of possible achievable
shapes. The latter allows the production of nanostructures in short time
and at low costs, offering the possibility to add desired chemical groups at
selected positions of the DNA chain, for further functionalization at the
single-molecule level.

1.3 DNA as Building Block of Self-assembled

Nanostructures
The initial step in formation of self-assembled DNA nanostructures is the
connection of distinct double helices together, creating so-called branched
DNA motifs or DNA tiles. The simplest branched motif is the Holliday
junction, a key intermediate in many types of genetic recombination and

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Nanotechnology and the Unique Role of DNA 9

Figure 1.3.   The Holliday junction in its distinct conformations. The stacked iso-I (a) and
iso-II (b) conformations predominate at modest and high salt concentrations. The open
conformation (c) is mainly observed at low salt concentration, and also potentially acts as
an intermediary of conformational changes between iso-I and II. Adapted with permission
from Ref. [67].

ds break repair mechanisms. The Holliday junction forms when two

homologous ds DNA molecules exchange their strands in one common
branch point [66, 67]. Thus, the main structural feature of a Holliday junc-
tion is the symmetry of its component sequences, which causes the branch
point to migrate (Figure 1.3).
Although this is of course essential for successful accomplishment of
its biological function, it is instead deleterious for construction purposes,
where a certain degree of structural rigidity of the constituent building
blocks is desired. For this reason, initial efforts were focused on the devel-
opment of an immobile Holliday junction, i.e. a motif which is designed
such to avoid any topological isomerization and is therefore incapable to
resolve into two distinct double-helical segments. This ambitious goal was
achieved in 1982 by Ned Seeman and signed the beginning of structural

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DNA nanotechnology [14]. In the following years, more complex DNA

motifs have been developed and different design strategies have evolved,
sometimes even against initial predictions, demonstrating that the field of
DNA design may still reserve many surprises. A more detailed description
of the current strategies and available software tools for design of DNA
nanostructures will be given in Chapter 2.
Once the building blocks have been designed, the next step is to
drive their self-assembly into the target structure. Different approaches
are nowadays available to reach this purpose and are mainly related to
the kind of “building units” chosen and the forces involved in their bind-
ing. Nevertheless, the underlying concept is equal in all cases: the
entropic loss that occurs when a single element moves from an
“unbound” state in solution to a “bound” state in the growing structure
must be compensated by a gain in the energy of binding, thus favoring
the self-assembly process. Intuitively, inter-unit recognition forces
should be strong enough to ensure specificity, and therefore guide the
system to a state of minimal energy, but sufficiently weak to enable
reversibility of binding, such that incorrect bonds can disrupt. In this
way, error-check and self-correction mechanisms can take place [68,
69]. If self-recognition interactions are too strong, the thermodynamic
forces driving the process to the optimal bound state may be counter-
acted by the slowness of escaping from misbound structures, thus lead-
ing to kinetic trapping (Figure 1.4(a)).
Another advantage in using DNA as a construction material is the
large variety of forces, which can be exploited to link DNA pieces
together. Indeed, besides canonical and non-canonical hybridization
forces (Figure 1.4(b)), base stacking offers another possibility to improve
the binding force, particularly at the blunt ends of larger DNA shapes
(Figure 1.4(c)) [70]. In this way, hierarchical self-assembly, i.e. the asso-
ciation of DNA units into higher-order architectures may be designed,
leading in some cases even to macroscopic structures. Interactions
between DNA units can be also directionally driven taking advantage of
geometric factors, like the shape-complementarity of the binding surfaces
(Figure 1.4(d)) [71]. Finally, long-range assemblies can also be favored
by confining the random Brownian motion of the constituents units to a
limited space, helping them to meet and bind [72–74]. Both approaches,

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Nanotechnology and the Unique Role of DNA 11

Figure 1.4.   The main forces driving the self-assembly of DNA nanostructures. (a) A
representative energy landscape of self-assembling DNA structures, showing that the step-
wise hierarchical association of single building units may follow different paths. From the
one side, short-life intermediate species may form which are capable to self-correct into
the target highly-ordered structure. In other cases, the unit-to-unit interactions may be too
strong to be counteracted, terminating the process into a kinetic trap, from which the ill
structure cannot easily escape. Unit-to-unit interactions in DNA self-assembly are mainly
hydrogen bonds between complementary bases (b). Other forces include base stacking
at blunt end helices (c) or shape complementarity (d). Adapted with permission from
Ref. [75].

i.e. shape-complementarity and template-assisted growth, may be viewed

as a strategy to restrict the number of isoenergetic states, eventually lead-
ing to a narrower energy profile with a uniquely defined minimum
(Figure 1.4(a)) [75].

1.4  Functionalization of DNA Nanostructures

Until now we have regarded the DNA molecule as the building unit of
self-assembled nanostructures. However, to make such structures useful

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for realistic applications, chemical functionalities must be added. This can

be done in two ways: from the one side, exploiting well-defined oligonu-
cleotide sequences as substrates of highly specific DNA-modifying
enzymes. From the other side, bringing desired DNA-tagged molecules at
selected positions of the nanostructured template through hybridization
with complementary single-stranded protruding arms. In both cases, the
DNA acts as a recognition motif either towards proteins or complemen-
tary DNA-tagged molecules. Specific examples of each strategy will be
reported below.

1.4.1  DNA as a recognition motif

In the past few years, Sugiyma and coworkers developed a DNA nanochip
for direct analysis of DNA-binding events. At this purpose, they immobi-
lized DNA recognition elements in the central cavity of a frame-shaped
nanostructure and analyzed binding or processing events at the single
molecule level, mainly using high-speed atomic force microscopy. In this
way they could control for example the tension of the immobilized
dsDNA substrate and thus demonstrate the importance of DNA-strand
relaxation and bending in base-excision repair mechanisms [76]. Similarly,
by tethering a Holliday junction intermediate into the DNA frame in dif-
ferent connection patterns, they found that the topological state of the
initial DNA substrate dictates the outcome of the recombination process
(Figure 1.5(a)) [77, 78].
In another important class of nanostructures, the DNA motif is not
metabolized or processed, but simply recognized by the incoming pro-
teins, thus serving as a molecular tag, for example, for targeting of tran-
scription factors [29] or site-specific positioning of DNA-binding protein
adaptors [79, 80]. A large family of functional DNA nanostructures relies
on the use of DNA aptamers for biosensing applications. One of the most
relevant studies of this kind was done by Yan and coworkers [81], who
used a DNA platform to anchor two thrombin-binding aptamers targeting
opposite sides of the same molecule (Figure 1.5(b)). By varying the dis-
tance between the two ligands, they could demonstrate optimal bivalent
binding at about 5.3 nm. This study represents the first example of using
the spatial addressability of self-assembled DNA nanoscaffolds to control

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Nanotechnology and the Unique Role of DNA 13

Figure 1.5.   Examples of DNA nanostructures carrying additional DNA sequences as

specific recognition motifs for DNA processing enzymes (a) and multivalent protein bind-
ing (b). DNA aptamers have been also used to actuate dynamic transformations (c), from
a locked status (aptamer–complement binding) to an unlocked status (aptamer–protein
binding). Adapted with permission from Refs. [32, 77, 81].

multi-component bimolecular interactions and to visualize them at a

­single-molecule level.
In other applications, DNA aptamers have been used to trigger
dynamic transformations (Figure 1.5(c)). The principle behind the
mechanical transformation is rather simple: two parts of the same con-
struct are initially connected by an aptamer and its complementary strand.
Addition of an aptamer-specific target induces the folding of the aptamer
and displaces it from the duplex, thus unlocking the two parts previously
bound together. This strategy has been used for the generation of mecha-
nochemical sensing platforms [82], aptamer-encoded logic gates [32] or
for controlled release of encapsulated cargo from a DNA cage [30].

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1.4.2  Synthesis of DNA–protein conjugates

As mentioned above, besides employing DNA as a recognition motif for
sequence-specific binding proteins, functionalization of DNA nanostruc-
tures can be attained through hybridization of DNA-tagged components to
complementary strands protruding out of the nanoscaffold. Semisynthetic
DNA–protein conjugates are hybrid biomacromolecules that combine the
unique structure-directing properties of DNA with the almost unlimited
variety of functional proteins. Proteins have been tailored by billions of
years of evolution to perform highly specific functions, such as molecular
recognition, catalytic turnover, energy conversion or translocation of other
components across membranes. In this sense, DNA–protein conjugates
represent an ideal tool for adding a desired functionality to nanostructures.
Various methods for the coupling of synthetic DNA oligomers with pro-
teins have been developed, based on either covalent or non-covalent strat-
egies. The chemistry of DNA bioconjugation is too large to be treated here
in detail and extensive literature is available on the topic [44, 83]. In the
following, we will therefore give a brief survey of the most important
methods adopted for the synthesis of DNA–proteins conjugates and how
they have been used for functionalization of DNA nanostructures.

1.4.2.1  Covalent coupling

In the covalent coupling approach, a DNA strand is modified with a
chemical function, which is then covalently bound to the protein-of-
interest (POI). A typical approach is based on thiol chemistry between a
thiol-modified DNA and genetically engineered cysteine residues of
recombinant proteins, thus allowing to prepare DNA conjugates with
defined stoichiometry and regioselectivity of the coupling site [84].
However, while disulfide bonds are still amenable to cleavage under
reductive conditions, the use of crosslinkers bearing a maleimide func-
tionality, such as sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-
1-carboxylate (sulfoSMCC), enables irreversible coupling. SulfoSMCC is
a representative example of the broad class of heterobispecific crosslink-
ers, which are used to couple amino-reactive groups exposed on the
protein surface with thiol-modified DNA oligonucleotides [85]. If a
­

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Nanotechnology and the Unique Role of DNA 15

Figure 1.6.    Examples of covalent (a) and (b) and non-covalent (c) DNA–protein com-
plexes used to functionalize DNA nanostructures. Typically, DNA–protein conjugates are
obtained by linking the two components by heterobifunctional crosslinkers (a). The con-
jugates are then attached to the nanostructure through hybridization with complementary
strands. Other methods instead rely on the covalent binding of protein tags to suicide
ligands previously tethered onto the nanostructure (b). Finally, the biotin–STV interaction
is the most widely used non-covalent binding for decoration of DNA nanostructure sur-
faces (c). Scale bars are 100 nm. Adapted with permission from Refs. [26, 28, 89].

site-specific linkage is required and the POI contains accessible cysteine

residues, the coupling order can be reversed, using amino-modified oligo-
nucleotides [86]. The use of heterocrosslinkers is still one of the most
largely employed methods for attachment of selected proteins to DNA
nanostructures (Figure 1.6(a)) [28].
However, in many cases, it is not possible to engineer a single chemi-
cally accessible cysteine into the POI. Therefore, coupling methods are
needed, which are orthogonal to the variety of functional groups in native
proteins. A typical example is given by the self-labeling proteins

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16 E.-C. Schöneweiß, A. Jaekel and B. Saccà

“Snap-tag” [87] and “HaloTag” [88] that specifically bind to their suicide
ligands benzylguanin (BG) and chlorohexane (CH), respectively. Both
Snap- and HaloTag have been genetically fused to POIs and subsequently
reacted with BG- or CH-modified DNA oligonucleotides, allowing for the
site-selective decoration of DNA nanostructures with multiple different
proteins (Figure 1.6(b)) [89, 90].

1.4.2.2  Non-covalent coupling

Probably the most widely employed non-covalent interaction between a
DNA strand and a protein takes advantage of the remarkable biomolecular
recognition of D-biotin (vitamin H) by the homotetrameric proteins avidin
or streptavidin (STV). Due to the enormous high affinity constant of the
biotin-(strept)avidin interaction (Kd = 10-15 M), the extreme chemical and
thermal stability of the STV, and the commercial availability of bioti-
nylated oligonucleotides, biotin–STV coupling has frequently been
applied in structural DNA nanotechnology, either as a proof-of-principle
for protein binding or as topographical marker for atomic force micros-
copy imaging (Figure 1.6(c)) [91]. Other affinity tags, such as the chromo-
phore fluorescein (Fsc), can also be coupled to DNA oligonucleotides. Fsc
can be used as a hapten to specifically bind immunoglobulin G (IgG)
antibodies raised against it. For example, Mao and colleagues have used
Fsc-derivatized oligonucleotides to assemble a hapten-modified DNA
nanoarray, which was then used as template to assemble antibodies into
high density arrays with a defined pitch of about 20 nm [26].

1.5  Dynamic DNA Motifs

The ultimate goal of DNA nanotechnology is the finest possible level of
control over the spatial and temporal structure of matter. Thus, besides
the use of DNA as a static framework to control the spatial arrangement
of molecules, the programmability of nucleic acids structures can be
conveniently employed to develop dynamic systems, whose interconver-
sion between distinct species occurs in a predictable manner [49]. The
first switchable molecular machine has been pioneered again by Ned
Seeman and coauthors in 1999 [92]. In their work, they describe the

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Nanotechnology and the Unique Role of DNA 17

Open

–
F
F F

Z–B B–Z

F
–
F

(a) –
F

a) b)
F
Closed
Walker
+A1
(b)
5′ Capture leg

8–17 DNA enzyme leg

3′
Track 3′ 3′
+A2
d) c)
Waste
+D1 rA

dA

25 nm

(c) (d)

Figure 1.7.    Examples of dynamic DNA devices, using the metal-induced B–Z transition
of the DNA helix (a) or the well-established single-strand displacement mechanism
(b). This latter has been used to create DNA-walkers, moving on 1D (c) or 2D (d) tracks
along prescriptive directions. Adapted with permission from Refs. [93, 94, 97, 100].

realization of a supramolecular device consisting of two rigid double-

crossover (DX) molecules connected by a short double-helical linker
(Figure 1.7(a)). The screw motion of the device was triggered by the
addition or removal of cobalt ions, which induced the transition from the
B- to the Z-form of DNA. The operation has been monitored by fluores-
cence resonance energy transfer spectroscopy, measuring the relative
proximity of two dye molecules attached to the free ends of the DX
molecules.
One year later, Yurke and coworkers published the first DNA machine
powered by DNA itself [93], thus establishing the basics of dynamic DNA

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18 E.-C. Schöneweiß, A. Jaekel and B. Saccà

nanotechnology. They reported the construction of a DNA device in which

the DNA was used not only as a structural material, but also as a “fuel”
(Figure 1.7(b)). The functioning of the machine was based on the predict-
able conformational transition between different species, driven by forma-
tion of thermodynamically more favored duplexes through a mechanism
of strand invasion and single-strand displacement. In the following years,
this principle has been successfully employed to develop bioinspired
DNA-nanomotors [52] and bioinformatic tools for emulating complex
reaction circuits [54]. Recently, Kuzuya and coauthors also demonstrated
that the structural reconfiguration of large DNA origami can be precisely
controlled and directed to one of three different states [94], thus suggest-
ing that more complex systems can be designed, in which the interplay
between structural and dynamic properties of the DNA molecule offers
enormous potential for future applications.
The controlled nanomechanical actuation provided by strand dis-
placement was also used to construct molecular devices that could con-
tinuously move along a predefined trajectory rather than switching
between a limited number of fixed configurations (Figures 1.7(c) and
1.7(d)). In this way, so-called DNA walkers have been produced, which
could move directionally along a DNA track, taking one step with every
input added [95–99].
Many of these devices, however, require the addition of external
input strands for continued operation and suffer from “poisoning” of the
system due to formation of so-called duplex waste. As in macroscopic
man-made machines, the efficiency of functioning is therefore limited.
With the purpose to overcome this drawback, scientists developed alter-
native strategies, such as the single-strand displacement cascades. In
these systems, the output of a reaction serves as the input to a down-
stream reaction, thus allowing for the engineering of complex autono-
mous devices [100, 101].
Finally, recent works have demonstrated the importance of kinetic
control of strand displacement for the programmability of autonomous
molecular machinery and molecular computation [102]. This can be
achieved by insertion of mismatched bps, thus allowing to tune the energy
landscape of the reaction in a predictable way. A remarkable example of
the extreme degree of control achievable using DNA nanotechnology

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Nanotechnology and the Unique Role of DNA 19

tools is represented by the work of Winfree and coauthors [103]. There,

they showed how two well-defined frameworks, i.e. DNA tile self-­
assembly and DNA strand-displacement circuits, can be integrated to
provide programmable kinetic control of self-assembly.
Thus, the future of DNA nanotechnology is without any doubt bright:
integrating more complex structures with dynamic circuits one can envi-
sion to achieve a precise spatial and temporal organization of matter with
a resolution of only few nanometers.

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Chapter 2
y.

DNA as Building Material

at the Nanoscale: From Concepts
to Software-aided Design

Wolfgang Pfeifer*, Georg Homa*, Giuseppe Arrabito†

and Barbara Saccà*,‡

*Centre for Medical Biotechnology (ZMB)

Universitätstr. 2 of Duisburg-Essen
45117 Essen, Germany
†
Department of Physics and Chemistry
University of Palermo
Viale delle Scienze, Parco d’Orleans II, 90128 Palermo, Italy
‡
barbara.sacca@uni-due.de
by

Chapter 2 is the most technical chapter of the book, since it deals with the
different approaches and strategies allowing for design of DNA nano-
structures such as tile-to-tile assembly or scaffold based design. The
reader will also find information about DNA aptamers design for applica-
tions in analytical chemistry.

27

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28 W. Pfeifer et al.

2.1  Basic Concepts of Design

Structural DNA nanotechnology is an example of how scientific original-
ity and human imagination can bring simple natural rules to such a high
level of sophistication to result into the founding of a new field of science.
Undoubtedly, the driving force of such a progress is DNA design. Since
the pioneering work of Ned Seeman in the early 1980s [1], multiple
design strategies have been developed. Despite all rely on the Watson–
y.

Crick base-pairing rule, they explore different ways for folding DNA into
complex topologies.
The main reason for the rapid assessment of distinct design approaches
is the predictability of the DNA self-recognition interactions, which has a
remarkable consequence: once a sequence is given, its complementary
sequence is uniquely defined. In this sense, the design of a single double
helix is a rather simple exercise. The task however becomes more difficult
when the purpose is to generate a pool of DNA sequences, which self-
assemble into distinct linear duplexes. In this case, indeed, the availability
of only four bases gives rise to sequence subsets that are inevitably identi-
cal. To bypass this problem, these subsets should be as short and rare as
possible, such that the selected sequences will result maximally different.
This means that the probability of each strand to hybridize exclusively
with its complement will become maximal [2–4]. Fortunately, software
tools are nowadays available, which basically “translate” this sequence
diversity into a numerical quantity, thus allowing for ranking the sequence
candidates according to a desired property [5].
The next level of design difficulty appears when distinct DNA helices
must be designed, such to interlace one another at one or more points
by

(called junctions) forming intricate shapes of higher structural order,

termed tiles [6]. In the design of DNA tiles, not only geometric factors and
topological constraints become important but also the thermodynamic and
kinetic factors that regulate the interconversion between the transient spe-
cies must be taken into account.
In the present chapter, we will describe how DNA tiles, and in gen-
eral, DNA building units, can be designed from scratch and which param-
eters can be varied to tune their properties, such as the mechanical

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DNA as Building Material at the Nanoscale 29

flexibility, the global curvature and the strength of the interactions with
other units to form extended structures. These latter represent the last level
of design challenge, where a subtle balance between different forces must
be achieved to ensure the successful building up of the desired assembly
[7]. To achieve this purpose, the interactions between the binding units
should be strong enough to be able to propagate over long distances but
sufficiently weak to enable unbinding and self-corrections mechanisms,
y.

thus preventing kinetic trapping of misfolded structures [8, 9]. Besides

base hybridization, other forces such as base stacking and shape comple-
mentarity [10–12], are often used to tune the interactions between the
single DNA units of the system, allowing to direct the self-assembly pro-
cess towards specific target shapes. In addition, experimental conditions
such as temperature, annealing rate, ionic strength as well as the presence
of surface supports or nucleation seeds can be optimized to improve the
self-assembly yield by simply increasing the probability of the units to
meet and bind [13–18].
It is therefore clear that the challenge of DNA design goes much
beyond the simple base-complementarity rule and that robust theoretical
models and software tools are necessary to enable the engineering and
analysis of DNA structures. Fortunately, many of the instruments needed
to achieve this goal are freely available in the internet and readily acces-
sible to the scientific community, thus notably contributing to the assess-
ment and rapid spread of this field of science.
In the following, we will give a brief introduction of the two main
approaches used until now for designing DNA nanostructures and will
describe the single strategies more in detail in the dedicated sections.
by

2.2  The Two “Schools” of DNA Design

Until now, the design of a DNA nanostructure can be performed following
one of two possible schools of thought: (i) the so-called multi-stranded (or
tile-based) approach, historically the first method established in the field
of DNA nanotechnology (Figure 2.1(a)) [1] and (ii) the scaffold-based
approach, commonly known as DNA origami, firstly reported by
Rothemund in 2006 (Figure 2.1(b)) [19].

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30 W. Pfeifer et al.
y.

Figure 2.1.    The two assembly strategies used in structural DNA nanotechnology. In the
multi-stranded (or tile-based) approach (a), oligonucleotides are designed to self-assemble
with each other into a well-defined branched DNA motif (also called “tile”). Hierarchical
assembly of such motifs into large (finite or infinite) periodic matter is achieved through
cohesion of sticky-ends. In the DNA-origami technique (b), a long single-stranded DNA
is folded into a finite-sized shape by the help of many shorter oligonucleotide sequences,
also called staple strands. Adapted with permission from Ref. [22].
by

The two strategies basically differ in the absence or presence of a long

single-stranded sequence, named scaffold, as the “main actor” of the
assembly process. This makes the design approaches, the attainable struc-
tures and experimental settings completely different and to some extent
even divergent. In the multi-stranded procedure, DNA sequences are
designed to self-assemble into branched motifs (also called tiles) of
defined geometry [6], following a rather strict principle of sequence-
symmetry minimization [5]. According to this principle, sequences

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DNA as Building Material at the Nanoscale 31

participating in the formation of the motif are chosen to be maximally

different from one another, such that competitive formation of alternative
secondary structures will be minimized. In a further step, those motifs are
used as building blocks for the construction of large periodic matter
through cohesion of their sticky-ends. This requires a high control over
the stoichiometry and purity of the constituent oligonucleotides, thus
resulting in error-prone and lengthy synthetic processes. In the DNA-
y.

origami technique, instead, a long single-stranded DNA sequence (termed

scaffold or template) is folded into a desired 2D or 3D shape by the help
of up to hundreds of shorter oligonucleotides (also named staples).
Although the first examples of such a scaffold-based approach were
reported by Yan et al. [20] and Shih and coworkers [21], neither of these
two papers had the impressive impact of Rothemund’s publication [19].
The high performance of the DNA-origami method is mainly attrib-
uted to the entropic advantage in using a single long scaffold strand for
folding [20]. In fact, since staple strands hybridize with a common scaf-
fold rather than with each other, their relative stoichiometric ratio is no
more relevant. Moreover, initial correct arrangement of the scaffold favors
the correct binding of the remaining staples, such that possibly existing
wrong or truncated sequences are easily displaced by strand invasion and
exchange mechanisms. Consequently, experimental errors and time of
synthesis are dramatically reduced because tight control over stoichiom-
etry and purity of the oligonucleotides is no longer necessary. This,
together with the capability to generate nanoobjects with complex shapes
of pre-defined dimensions and full molecular addressability, makes the
DNA origami a very robust and powerful tool for construction of DNA-
based architectures [22].
by

Recently, alternative strategies have been also reported, belonging

either to the multi-stranded or scaffold-based class. An example of tile-
based approach is the wave tile design (Figure 2.2(a)), which uses two
component strands interlaced back and forth to form flexible periodic
architectures [23]. Using multi-arms junctions with low symmetry, intri-
cate tessellation patterns based on Archimedean tiling have been also
produced (Figure 2.2(b)) [24]. Aperiodic structures are instead more eas-
ily achievable through the so-called single-stranded tile (SST) approach
(Figures 2.2(c) and 22.2(d)) that relies on the use of several hundreds of

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32 W. Pfeifer et al.
y.
by

Figure 2.2.    Examples of tile-based approaches. Whereas the weave tile design uses two
component strands interlaced back and forth into flexible periodic architectures (a) [23],
DNA branched junction of variable topological symmetry enable the formation of Platonic
or Archimedean tessellation patterns (b) [24]. Instead, the SST approach relies on the use
of several hundreds of distinct sequences designed to hybridize one another into simply
concatenated duplexes, thus giving rise to 2D (c) or 3D (d) structures with a high level of
complexity and addressability [25, 26]. Adapted with permission from Refs. [23–26].

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DNA as Building Material at the Nanoscale 33

distinct sequences designed to hybridize one another into concatenated

duplexes [25, 26]. This method enables the generation of more than 100
arbitrary shapes from modular combinations of several hundreds distinct
molecular pieces. Despite the low yield of the process and the challenging
design of the sequences, the sophistication of the structures attained and
their modular construction make this approach still unrivaled.
Using instead a scaffold routing algorithm based on graph theory [27]
y.

or wireframe patterns with controlled angles [28], net-like DNA shapes

can be produced which would be otherwise almost impossible to achieve
with other methods (see paragraph 1.4). Altogether, the diversity of cur-
rent design procedures and the frequent emergency of novel approaches
demonstrate the huge potential of DNA-guided self-assembly, where a
simple rule applied to four pieces can still offer many possibilities.

2.3  The Multi-stranded Approach

2.3.1  The first immobile junction designed
by SEQUIN
The first rationally designed DNA motif was an immobile Holliday junc-
tion [1], later optimized into the J1 motif (Figure 2.3) [5]. Contrarily to its
analog transient species naturally occurring in genetic replication and
recombination, the J1 junction was designed to avoid (or minimize) its
topological isomerization, i.e. its resolution into distinct double-helical
segments.
In this sense, the definition of immobile refers to the incapability of
the motif to undergo branch migration at the junction and is not correlated
by

to its structural properties. The J1 motif was designed with SEQUIN, a

program to assign nucleic acid SEQUences INteractively. More correctly,
the SEQUIN program was developed in order to design the prototype
motif of structural DNA nanotechnology and is still one of the most domi-
nant software tools for the design of immobile DNA junctions in
general.
The first step in designing a DNA junction is to define its molecular
connectivity. This implies specification of the number and chain length of
the arms, presence and position of the junction, single-stranded loops and

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34 W. Pfeifer et al.

I
100

CGCAATCC
GCGTTAGG

PERCENT JUNCTION
80
1 4
60
GCACGAGT TGATACCG
II IV
CGTGCTGA ACTATGGC
40
CCGAATGC
GGCTTACG

2 3 20 Fidelity = 0.9998
y.

Calc. Tm = 48ºC
0
–20 0 20 40 60 80
III
J1 TEMPERATURE
(a) (b)

Figure 2.3.    The optimized immobile DNA junction (J1). (a) The structure is composed
by four hexadecamers forming a cross-like structure with 8 bp long arms. The structure
has been designed with the SEQUIN program according to a sequence-symmetry minimi-
zation algorithm, which minimizes the probability of formation of alternative assemblies.
Some critons are indicated by dashed boxes. (b) Besides sequence symmetry rules, ener-
getic criteria (i.e. fidelity and melting profile) must be also taken into consideration for the
realization of an immobile junction. The J1 motif has been selected as the “best” candidate
of a four-arm junction, because presenting the highest calculated melting temperature
among those motifs with the highest fidelity and a sigmoidal (all-or-none) transition pro-
file. Figure (b) was adapted with permission from Ref. [5].

double-stranded linkages between the arms. In terms of SEQUIN, the

rank (R) of a structure is defined as the number of double-helical portions,
which directly emerge from the junction. Therefore, in the J1 motif of
by

Figure 2.3(a), the four-arms junction has rank R = 4. Another structural

parameter of the junction is the bend: i.e. the phosphodiester linkage,
which is flanked by bases paired to different strands. In the example
shown in Figure 2.3(a), the bend of strand 1 in the final structure is
flanked by one C and one T, respectively paired to one G (of strand 4) and
one A (of strand 2). Similarly, the other three strands also show one bend;
the target four-arm junction has therefore totally four bends. Each of the
four strands participating in the formation of the J1 motif is 16 bases long
and can be considered as composed of 13 consecutive and partially

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DNA as Building Material at the Nanoscale 35

overlapping four-base segments (highlighted by dashed boxes in strand 1

of Figure 2.3(a)). In the reported example, the first segment at the 5′ ter-
minus of strand 1 will be the CGCA tetramer, the second segment (trans-
lated only by one nucleobase position towards the 3′ direction) will be
therefore the GCAA sequence and so on, up to the last four-bases long
segment at the 3′ end of the same strand, which will be CACG. Each of
these segments is termed a criton. The number of different critons of
y.

length N generated by a four-bases code is 4N. Thus, the total number of

four-bases long critons (N = 4) will be 44 = 256. These are the different
subsequences that are in principle available for construction of the desired
junction. At this point a selection criterion must be defined in order to
reduce the pool of available sequences. In the SEQUIN program such
criterion is the minimization of the sequence symmetry. Basically, the
software tries to avoid base complementarity at undesired positions, thus
reducing the chances of formation of alternative associations. More in
detail, the program checks for the fulfillment of the following requisites
within all selected pairing regions: (i) each criton may appear only once
throughout the whole structure; (ii) the complement to any criton that
spans a bend must not be present in any strand; (iii) self-complementary
critons are not permitted; (iv) the same base pair at the junction can appear
only twice and, if so, must be located on adjacent arms. In the J1 motif of
Figure 2.3(a), the following base pairs are present in the central junction:
CG (north arm), TA (east), GC (south) and AT (west). These are the four
possible distinct combinations allowed, when considering the polarity of
each double-helical arm. For example, in the CG pair of the north arm, the
C occupies position 8 (counting from the 5′ terminus of strand 1) and G
occupies position 9 of the complementary strand 4. This base-pairing
by

configuration differs from the GC pair of the south arm, in which the two
bases exchange their relative position (G is now in position 8 and C in
position 9). This opposite polarity prevents the hybridization of the bases
located in opposite arms.
If a rule violation (e.g. use of a self-complementary criton) is neces-
sary because of additional requirements (e.g. the placement of a restriction
site) the user can choose to tolerate it.
In a further step, free-energy criteria are taken into account to ensure
reliable attainment of a stable branched motif at working temperature

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36 W. Pfeifer et al.

conditions. The program addresses this issue by adding two levels of

design control: (i) calculation of the fidelity of the junction and (ii) calcu-
lation and plotting of the melting curves. The fidelity (p) of a structure is
described as a probability function according to the Boltzmann’s
distribution:
-DGJ
e RT
p=
y.

,
Z

where DGJ is the free-energy of the desired junction, R is the gas con-
stant and Z is the partition function, that is, the sum of the energetic
contributions given by all competitive pairings represented by adjacent
sets of two base pairs. The fidelity of a junction can be calculated from
the DG° values reported in the literature for pairing of two-bases long
segments [29], assuming that the equilibrium constant for junction for-
mation (KJ = exp -DGJ/RT) is given by the weighted product of the bind-
ing constants of all two bp-subunits (i.e. KJ = b K1 K2 K2 …. Kn-1; where
b is the nucleation constant for initial strand hybridization and n is the
length of the complementary sequences (all values are intended at 25°C)
[5]. The junctions with the highest fidelity values are retained by the
program and further analyzed for their thermal stability. Theoretical
melting curves for the DNA junctions are then calculated assuming
equal initial strand concentration for all strands participating in tile
assembly. Melting curves are plotted as fraction of the junction formed
in function of the temperature. A well-designed junction should present
a sharp uniphasic melting profile with maximal melting temperature
(Figure 2.3(b)).
by

Experience shows that 100% stringent observation of the rules

described above is not always necessary and that a certain degree of flex-
ibility through introduction of manual changes from the user is often more
efficient. The accuracy of the SEQUIN algorithm for the design of DNA
motifs of desired geometry has been largely demonstrated by the success-
ful realization of DNA junctions of higher complexity, such as five, six,
eight and even twelve-arm junctions (Figures 2.4(a)–2.4(d)) [30, 31]. The
program has also been used for building motifs with multiple branch
points, such as the double-crossover (DX) molecules (Figures 2.4(e) and
2.4(f)) [32] or the 4 × 4 tile (Figure 2.4(g)) [33].

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DNA as Building Material at the Nanoscale 37

Single-junction motifs Multiple-junctions motifs

I

GGTTCAGCCGCAATCC
CCAAGTCGGCGTTAGG
(a) I (b) (e)
GGTTCAGCCGCAATCC
CCAAGTCGGCGTTAGG

II CC
A
GG TCT 1 5 TA
GG
TG CC
VI
TA CA TG AT
GA CG CG AC
GT TCC AC CAC
GC T
AG GCT A G
CCA 1 5 G V GC TG ATG
T
II GGT TCTCA GTG
TAG C
CGT CACATC
GA AC
AGA C
GTG GTCCG
CAG C TAC
TGA ATGGCA
2 GT
GC 6
GCG T GA CG AGGT
ACT AC CA TC CG
A
GC CT CA AG DAO

GGCTTACGGTGGTGCG
CCGAATGCCACCACGC
4 GA TG GC AC
2 CT GC TC TA
GG GAA
CT T

TT GAGT TG GG
CC
CT TG
TG AG
CA

AT
G
C AA
3 4 CC
TA CC
GC CG

III GC V (f)
CG AC
GT CA

3
GT CA
GA AG
AA TG

GG CG
GC TTC

TG C
CG
CG
y.

III IV
IV

(c) (d) DAE

I
II XII
I
GGCACAGCTATAATAACGCAATCC
GGATTGCGTTATTATAGCTGTGCC

(g)
GG
CC
GGTTCAGCCGCAATCC

GG
CCAAGTCGGCGTTAGG

CC
AA
TA
TA

TT
G
TT

TG
AA

AC
II VIII
AC

CA
GT

III
GC

XI
CT

CC
TG
AG

AT
CC G

AA

AT
CA TG

TA
TT

AC

GG
12
GG

C
CA

CT GC CG AT
TA TG

1 5
GC
TG

C
CT

1
TA

GT GA CA
TC
CT

CA
CA TA

CG TC
GA
GA
AG
AG

TG
GA

GC

AG TC CT
TT
CG
CG CG

GC

AA AT
GT TA
GC

CC

11
GT

CG

TC AA
TC
CA
CT
AA TG

2
AT
TG
CG

AT
TT
CT GA
GC

CG A
CT AG
AA
TC GT

AG
TA

GG
GA

TA
TG
AG

CC
TC

CT AT T AA
GG CA

2 AT
T

8
AC

CA
GA

CA
GC

CG
TC TT
CC
3 10
C

GG CG
GA

X
GCAAGGTTGACACTTCCGTGCTCA
VI IV
ACAATTACGAACCAACTTAGGACC
CGTTCTGAGCACGAGT TGATACCGTGTGTAGG
III GCAAGAGTGCTGCTCA ACTATGGCACACATCC
GGTCCTAAGTTGGTTCGTAATTGT
GG
TC
4 C GC
TGAGCACGGAACTGTCAACCTTGC
9 AT
GC
AT
AC AT AG
T
CC GC

TC

CG
TG CT TG
AC
GA

AG

CA
3 7 GA
TG CA

TG
GCAAATTCAAGTCACACCTGTACG
CGTACAGGTGTGACTTGAATTTGC
G

TA
GA TT

TT

GC
AC TA
A

AC
AA

CG
GCAGGTCGAGACTAGG

TG

5
TT AT

8
CGTCCAGCTCTGATCC

GT
CC CG
A A

GC
GC
CG
TA

AC
T

G
CG

TT
AC GA

CT AG
TA
AT
GC GG

CA
7
A
TC

GG 6 TA
AT

TA T
AG
TG CG
C

GA
CT
CA TG

T TA TC
AT

TA CG
AG

AA

CG
CA TA

CC
AC

TA
CC GT

GG CC
GT

4 6 GG
GT

TA
TC GG

CC
AC GCG

TT

IX
GG

CA
CA TC

GA
GC

V
G
AC
TC
GG C

TT
TA

AA
TA

GC
GG

IV VII
GC
CC

TC
CT

GA
AG

GG
CC

CC
GG

V
VI VIII
VII 4x4

Figure 2.4.    Branched DNA motifs containing one (a)–(d) or more (e)–(g) branch points.
DNA junctions containing 5 (a), 6 (b), 8 (c) and even 12 (d) branches have been designed
by SEQUIN and successfully obtained as single species. Note that increasing the number
of branches does not necessitate redesigning the whole tile: additional arms are intro-
duced into a pre-existent immobile junction in order to fulfill the required symmetry-
minimization rules. More complex motifs realized with SEQUIN include the DX tile with
an odd (DAO, e) or even (DAE, f) number of half-helical turns between the two crossovers
and the more intricate 4 × 4 tile (g), formed by the self-assembly of nine oligonucleotides
into four four-way junctions. Adapted with permission from Ref. [40].
by

Though being a very useful tool, SEQUIN still requires a significant

amount of manual operation in the sequence optimization process and
uncommon informatics skills. Therefore, alternative programs have been
developed in the past few years, aiming at reducing the manual input from
the user and attempting to provide user-friendly interfaces and graphical
representations of the output structures. A significant example is the
CANADA package [34], employed for the automated design of separate
and concatenated oligonucleotides for the DNA-directed immobilization
of proteins on a microarray [35, 36] and for the design of complex

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38 W. Pfeifer et al.

branched motifs [37]. Other relevant examples are GIDEON [38],

Uniquimer 3D [39] and NanoEngineer-1 (available free of charge at http://
www.nanoengineer-1.com). Those programs provide a virtual graphical
model of the DNA motif and are supplemented with elementary algo-
rithms for minimization of the energy of the construct [39]. Through an
iterative relaxation process, the structural elements of the motif are fixed
into a configuration of minimal strain, thus obtaining a rough estimate of
y.

the final most probable arrangement of helical segments in the target

structure. In this way, it is possible for example to visualize the intrinsic
curvature of a single tile, which must be taken into consideration when
binding multiple tiles together, as explained in the next section.

2.3.2  Tile-to-tile assembly

The initial vision and final goal of structural DNA nanotechnology is to
construct molecular architectures of desired geometrical features.
Therefore, once single junctions are realized, the next step is to connect
them into networks. This is achieved through sticky-ends cohesion, i.e.
through hybridization of complementary single-stranded segments pro-
truding from the 5′ or 3′ termini of junction’s arms. Of course, also at this
stage certain design criteria must be observed.
Two main parameters define the geometry of tile-to-tile assemblies:
(i) the angles between the double-helical segments emerging from the
crossovers and (ii) the distance between joined crossovers. The angles at
the central junction of a tile are normally defined by the length of the
single-stranded segments inserted between adjacent arms and from the
total number of arms among which the mechanical stress is distributed.
by

For example, inserting three thymine bases between two adjacent arms of
a three-arms junction one obtains a motif with a C3 rotational symmetry,
which tiles the plane into a regular hexagonal pattern (Figure 2.5(a)).
Such a tiling is also referred to as 6.6.6, indicating that at each junc-
tion three hexagons meet at their vertices tiling the plane into three identi-
cal angles of 120° [41]. By lowering the sequence symmetry of the
system, distinct Archimedean tiling can be obtained [24]. Starting for
example from the same three-arms junction, the number of thymine loops
at the junction can be varied such to result into one angle of 90° and two

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DNA as Building Material at the Nanoscale 39

y.

Figure 2.5.    Platonic and Archimedean tilings of the plane. Using a sequence symmetric
three-arms junction (with three T3 loops), the rotational C3 symmetry leads to identical
120° angles between the tile arms, which in turn allows for the formation of platonic tilings
of the kind 6.6.6 (a). Lowering the sequence symmetry of the tile (still keeping the same
number of T loops), enables to tune the tile angles such to get 4.8.8 Archimedean patterns
(b). In a similar way, a 4 × 4 tile of sequence and topological C4 symmetry (with four
identical T4 loops) results in the formation of 4.4.4.4 tessellations (c). An analogous tile of
lower symmetry (two opposite T3 and T5 loops) (d) leads instead to 3.6.3.6 tilings of the
plane.

angles of 135°. Such a motif will tile the plane into regular squares and
octagons in a 4.8.8 pattern (Figure 2.5(b)). Similarly, using a 4 × 4 tile,
regular square-like patterns can be achieved (4.4.4.4, Figure 2.5(c)) or
by

3.6.3.6 tessellations (Figure 2.5(d)).

Of course, one should consider that the torsional stress at the central
junction imposed by the connection between adjacent arms gives rise to
dihedral rather than plane angles and that this will finally end up into a
precise tile curvature. Such an intrinsic curvature is of fundamental impor-
tance for the final architecture of the assembly product, which can assume
completely different shapes depending on the second parameter of tile-to-
tile association, i.e. the distance between adjacent crossovers. Considering
n as the number of half-helical turns between joined crossovers, an even

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40 W. Pfeifer et al.
y.

Figure 2.6.   Tile-to-tile assembly. Graphical model of 4 × 4 tile extended lattices

obtained by the Uniquimer 3D software. Visual models were obtained after an iterative
process of energy minimization, which leads to the conformation of minimal strain. As the
4  ×  4 tile is not a planar structure, sticky-ends cohesion between adjacent tiles leads to
formation of large periodic structures, whose architecture depends on the number of half-
helical turns between adjacent junctions. Whereas an even number of half-helical turns
between interconnected junctions results in accumulation of tile curvatures and formation
of tubular structures (a), an odd number of half-helical turns results in curvature compen-
sation (also called corrugation), thus yielding to quasi-planar lattices (b). Adapted with
permission from Ref. [40].

multiple of half-helical turns (2n, Figure 2.6(a)) will result in accumula-

tion of the intrinsic curvature of the tile and formation of a closed assem-
bly [42]. On the contrary, cohesion of adjacent tiles through an odd
multiple of half-helical turns (2n + 1; Figure 2.6(b)) will cause them to
face up and down alternately (according to a so-called “corrugation strat-
by

egy”) with formation of a quasi-planar array [33].

Finally, the thermal stability of tile-to-tile interaction is directly
related to the strength of sticky-ends cohesion. This can be typically
enhanced by increasing the CG ratio and/or the length of the complemen-
tary DNA segments. On the other hand, excessive hybridization forces
should be avoided, as this might result in the trapping of misfolded struc-
tures thus reducing formation of the desired network. As a rule of thumb,
three to five nucleobases long sticky-ends are normally sufficient to
ensure specificity and stability of the newly formed double-helical “bond,”
still enabling self-correction mechanisms to take place.

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DNA as Building Material at the Nanoscale 41

2.3.3  Platonic and Archimedean tilings

Tilings or tessellations are ways of filling up the plane with tiles [43].
Normally, tilings satisfy the following conditions: (i) the tiles are regular
polygons, (ii) the tiling is edge-to-edge: this means that two tiles inter-
sect along a common edge, only at a common vertex or not at all; (iii) all
the vertices are of the same type: this means that the same types of poly-
gons meet in the same order (ignoring orientation) at each vertex. There
y.

are only three tilings that use only one type of tiles. These are called
Platonic or regular tilings. On the other hand, there are eight tilings that
use more than one type of tiles. They are called Archimedean or semi-
regular tilings [44]. To indicate such geometric patterns, the vertex con-
figuration is given. This is a sequence of numbers representing the
number of sides of the faces going around the vertex. For example, the
notation 4.8.8 describes a vertex that has three faces around it, namely a
square and two octagons. Several theoretical frameworks have been
developed for predicting the outcome of self-assembly processes leading
to tiling of the plane [45]. In the field of DNA self-assembly, Yan and
coworkers recently described a design strategy that enables formation of
Archimedean tilings by using only one type of tile with lower symmetry
(Figure 2.2(b)), thus enlarging the toolbox of DNA tile-based self-­
assembly and expanding the complexity of structures attainable by DNA-
based tessellation [24].

2.3.4  Single-stranded tiles

Tile-based assembly normally leads to formation of periodic structures of
by

unlimited size. In 2012, Yin and coworkers developed a design approach

to construct finite yet complex shapes from a large number (hundreds to
even one thousand) of uniquely addressable tiles [25]. The method relies
on the simplest form of tile: a single-stranded DNA (SST) constituted by
42 bases divided into four domains, each binding to four local neighboring
tiles during self-assembly (Figure 2.2(c)). Considering each SST as a
pixel, the method is based on the generation of a master strand collection
that corresponds to a 310-pixel canvas, from which appropriate subsets of
strands are selected to achieve a desired 2D shape. In this way, SST
assembly provides a simple and modular framework for constructing

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42 W. Pfeifer et al.

aperiodic nanostructures of complex addressability. The same concept has

been extended to 3D shapes, using so-called “DNA-bricks” as building
blocks of 3D canvas (Figure 2.2(d)) [26]. Each brick is constituted by
32-nuleotide long strands, which bind to four neighboring modular com-
ponents through 8-bp interactions. By selecting subsets of bricks from the
molecular canvas, the authors constructed a series of distinct 3D shapes
with sophisticated surface features as well as intricate interior cavities and
y.

tunnels.

2.4  Scaffold-based Designs

An extraordinary breakthrough in the construction of nanometer sized
DNA objects occurred in 2006 with the introduction of the scaffolded
DNA-origami method by Rothemund [19] (for reviews on this subject, see
Ref. [46]). Similarly to the Japanese art of paper folding, the DNA-
origami technique folds a long single-stranded DNA scaffold into a
desired shape by the help of hundreds of short oligonucleotides, called
staple strands. The arrangement of crossovers between the connected heli-
ces of a DNA origami defines the shape of the structure. This can be
planar, with helical axes arranged in an antiparallel fashion along single
monolayers, or space-filled, with helical axes oriented one another into a
hexagonal or square pattern (Figure 2.7).

2.4.1  Monolayer DNA origami

In the original work of Rothemund [19], several planar origami structures
were produced, ranging from simple rectangular shapes to more complex
by

forms, such as stars, triangles, as well as non-geometric figures as for

example smiley faces (Figure 2.7(a), left side). These objects were gener-
ated by following a common design rule: every helix within the structure
is connected to two neighboring helices by a regular pattern of crossovers
interspaced by 1.5 helical turns, which for a B-type DNA, correspond to
about 16 base pairs. This register of crossovers generates interhelical con-
nections every 180° thus leading to a single layer of helices arranged into
a planar sheet.

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DNA as Building Material at the Nanoscale 43

y.

Figure 2.7.    Schematic representation of crossover patterns and thus-generated lattices

used for the construction of 2D and 3D DNA-origami structures. DNA helices are indi-
cated by circles and are viewed along their central axis. Interhelical crossovers between a
reference helix (grey circle) and its neighboring helices (white circles) are indicated as
black arrows. The number of base pairs between consecutive crossovers along the same
helical axis is given, as well as the resulting 2D or 3D arrangement of the helices. Single-
layer (a) and (b) and multi-layer (c)–(g) DNA-origami structures can be generated by
suitable engineering of crossovers, thus leading to a large variety of shapes, including
by

vacant, filled, twisted and curved objects. Adapted with permission from Ref. [22].

The same design principle can also be used to generate 3D structures.

To this end, an origami is assembled which contains individual 2D sheets.
Those sheets are then connected at specific angles by an additional set
of  crossovers between interfacing helices at the edges (Figure 2.7(a),
right side). This generates 3D objects with an internal cavity, such as

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44 W. Pfeifer et al.

prisms-like structures with three, four or six faces [47], or closed polyhe-
dra, such as a tetrahedron [48] or cubes [49, 50].
Monolayer origami objects with complex curvatures were also pro-
duced by bending DNA helices along their central axis and linking them
together by a network of latitudinal and longitudinal crossovers to pro-
duce 2D and 3D structures, respectively (Figure 2.7(b)) [51].
Contrary to previously reported methods, this network of crossovers
y.

does not strictly obey the rule of 10.5 bp/turn, but rather permits a certain
degree of structural flexibility (from 9 to 11 bp/turn), which allows for a
more accurate tuning of the DNA curvature. This in turns gives access to
shapes such as planar concentric rings, spheres and hemispheres, which
are not reachable by conventional origami methods.

2.4.2  Space-filled DNA origami

One major limitation of single-layer DNA origami is their relatively weak
resistance to mechanical stress. To address this problem, more rigid 3D
DNA objects have been developed by either packing multiple helices into
a space-filling structure [52] or taking advantage of tensegrity rules
(Figure 2.7(c)) [53]. Multi-layer DNA-origami structures are densely
packed arrays of antiparallel helices interconnected through a defined 3D
arrangement of crossovers. The register of such crossovers, that is their
relative angular displacement with respect to the axis of the helix, defines
the way adjacent helices are interconnected and therefore determines the
geometry of the basic building block. The first example of multi-layer
DNA origami was reported by Shih and coworkers [52]. In their design
strategy, every helix is connected to three adjacent helices by crossovers
by

relatively oriented at 120°, spaced by 7 base pairs along the helical axis.
The resulting superstructure has therefore a hexagonal cross section simi-
lar to a honeycomb lattice (Figure 2.7(c), upper panel). The generality of
the method was demonstrated by successful formation of a series of 3D
shapes resembling for example a monolith or a square nut. Following the
same design principle, DNA objects were built with a rectangular [54] or
closed-packed hexagonal cross-section [55] enabling construction of flat-
ter surfaces, smaller cavities and denser structures (Figure 2.7(d)). Shortly
later, Dietz et al. were able to engineer supertwisted or bended structures

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DNA as Building Material at the Nanoscale 45

through deletion or addition of a distinct number of base pairs within heli-

cal arrays (Figures 2.7(e)–2.7(g)) [56].
In a different approach, Liedl and coworkers introduced mechanical
stability into 3D DNA objects taking advantage of the tensegrity concept
(Figure 2.7(c) lower panel) [53]. The term “tensegrity” stays for “ten-
sional integrity” and indicates geometrical integrity upon tension. This
architectural principle is often applied in macroscopic buildings, where
y.

geometric objects comprised of stiff sticks (“struts”) are connected by

flexible linkers (“tendons”). Since struts push outward and tendons pull
inward, the balance between the two forces leads to stable and mechani-
cally rigid structures. As demonstrated by Liedl et al. [53], 3D tensegrity
prisms can be constructed which are composed of rigid DNA bundles of
three to six helices, working as compressive-resistant struts, held in place
by ssDNA segments, which function as entropic spring tendons.
It should be stressed that such a rapid development of the DNA-
origami technology and the realization of sophisticated structures would
have not been equally effective without the help of adequate design pro-
grams. Fortunately, software solutions for designing 2D [57] and 3D [58]
origami are in continuous evolution and made available on a public
domain. Semi-automated design tools such as caDNAno (http://cadnano
.org) enormously reduce the time required for generating a new structure
and, more important, drastically lower the probability of human errors
which may occur in a completely manual process. In addition, programs
such as CanDo (http://cando-dna-origami.org) are available for the analy-
sis of the designed structures enabling to predict for example their thermal
fluctuations and geometrical properties. These programs are also con-
stantly upgraded with new features and functions, which make possible to
by

apply variations on design strategies in an easier way [11], thus notably

boosting the potential of the technology.
Finally, alternative strategies have emerged that seek to scale up the
dimensions of DNA nanostructures thus trying to fill up the gap between
the nano- and macroscopic world in a rationally designed manner.
Examples of such strategies include the use of so-called “short scaffold
parity strands” [59] or a set of different nucleobase-specific forces, shape-
complementarity and template-related effects to connect origami shapes
together into large hierarchical assemblies [7].

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46 W. Pfeifer et al.

2.4.3  Wireframe DNA origami

In the original DNA-origami design, the scaffold strand and correspond-
ing double helices are organized into parallel, raster-fill patterns. This
results into tightly packed structures, constituted by domains of adjacent
antiparallel helices joined by DX motifs. Few years later, Yan and cowork-
ers firstly faced the challenge to construct scaffold-based structures of
wireframe geometry (Figure 2.8).
y.

Wireframe structures are realized by linking multi-arm junction units

together, each one containing 2 to 12 arms, thus allowing for the non-
parallel alignment of DNA helices. This results in the formation of 2D
net-like surfaces with various sizes and shaped cavities as well as 3D
polyhedrons, curved solids and multi-layered frameworks.
Using a four-arms junction as the basic repetitive unit, Yan and cow-
orkers succeeded in folding the scaffold into planar gridiron architectures
(Figure 2.8(a)) [60]. Experimental observations revealed the formation of
rhomboid rather than square structures, due to the flexibility of the motif
and its relaxed conformation into a right-handed twist with a 60° torsion
angle. The strategy was extended to the third dimension either by control-
ling the curvature of the single motifs to get distorted shapes (Figure
2.8(b)), or by stacking or intertwining multiple layers of 2D gridiron lat-
tices at selected connection points (Figure 2.8(c)).
Multi-layered DNA frameworks with well-controlled geometry, sus-
tained mechanical stability and tunable cavity size have been lately cre-
ated by a novel layered-crossover motif (named as LX) [61]. The LX
motif is based on the DX motif with the difference that the DNA helices
connected by the crossovers are not belonging to the same plane, but to
by

two parallel layers (Figure 2.8(d)). In this way, DNA helices can be
aligned in a non-parallel arrangement through stacks of layers, thus
enriching the toolbox of structural DNA nanotechnology (Figure 2.8(e)).
The same group demonstrated the versatility of the idea by implementing
arbitrarily designed connections between selected multi-arms junctions in
a 2D and 3D space (Figure 2.8(f)) [28].
The design relies on a four-step process. In the first step, all connec-
tions between the vertices of the wireframe structure are converted into
double lines. In the second step, lines are looped and bridged such to

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DNA as Building Material at the Nanoscale 47

y.
by

Figure 2.8.   Wireframe-based DNA origami designs. Using a Holliday junction as build-
ing unit, 2D (a), curved (b) and lattice like (c) gridiron structures can be obtained [60].
Starting instead from DX motifs spanning two adjacent layers (LX motifs, d), rigid multi-
layers frameworks can be produced (e) [61]. The same principle applied to multi-arms
junctions with arbitrarily designed connections allows constructing sophisticated 2D and
3D patterns (f) [28]. A latest strategy instead, based on the graph theory, folds the scaffold
according to a triangulation pattern, eventually leading to formation of polyhedral meshes
(g) [27]. Adapted with permission from Refs. [27, 28, 60, 61].

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48 W. Pfeifer et al.

ensure the travelling of the scaffold strand through all vertices only
once, enabling also a circular path. In a third step, the complementary
staple strands are added to the scaffold lines to generate DX tiles spaced
by a full number of helical turns. This is done to minimize strain and
allow the scaffold to follow the predesigned path. Final step is to adjust
the angles at the vertices. This is achieved by inserting a Tn loop of
appropriate length into the staple strands surrounding the vertex and
y.

leaving a certain number of nucleobases in the scaffold — opposite to

the Tn loop — as unpaired. In this way, each vertex is provided with a
certain degree of structural flexibility such that the arms are allowed to
bend and position in the most favorable manner, eventually generating
the desired set of angles. The method has been applied for the construc-
tion of simple platonic tilings, curved patterns as well as intricate and
arbitrary shapes in the form of three flowers and a bird (Figure 2.8(f)).
In addition, 3D architectures of Archimedean symmetry have been also
produced using Schlegel diagrams [62] to generate the looping path of
the scaffold.
Similar polyhedral meshes were obtained by Högberg and cowork-
ers using a completely different design approach [27], based on the
“Chinese postman tour” [63] problem in graph theory (Figure 2.8(g)).
This design strategy relies on three main principles: (i) the meshes of the
structure are triangulated in order to optimize structural rigidity; (ii) the
edges of the structure are constituted by single double-helical segments
joined one another at common vertices and (iii) these latter should not
cross, that is, the scaffold should pass only once through all vertices.
Also in this case, the overall design process is split into four steps,
which can be run with the help of a software tool specifically created by
by

the authors (vHelix, available free of charge at http://www.vhelix.net).

In a first step, the 3D polygonal mesh is created using the 3D graphical
interface. In a second step, the routing of the scaffold through all the
edges of the mesh is generated. Third, the least strained DNA helix
arrangement is determined and finally, fine-tuning of the design through
a “relaxation algorithm” and assignment of the staple strands is per-
formed. In this way, different polygonal 3D meshes were generated,
including an icosahedron, a nicked torus, a bottle and a version of the
Stanford bunny.

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DNA as Building Material at the Nanoscale 49

2.5  DNA Aptamers: From Basics to Applications

Aptamers (from the Latin aptus — fit, and Greek meros — part) are
defined as DNA or RNA molecules — typically <100-mer, that bind to a
specific target molecule of bioanalytical significance with high affinity
and specificity. Aptamers are evolved by an evolutionary process, defined
as systematic evolution of ligands by exponential enrichment (SELEX ) in
which sequences with a certain conformation capable of binding to the
y.

target of interest emerge and dominate the oligonucleotides pool. The

SELEX strategy was firstly described in 1990 by the laboratories of Gold
and Szostak [64, 65]. The Gold lab used the term SELEX for their process
of selecting RNA ligands against T4 DNA polymerase. On the other hand,
the Szostak lab defined the term “in vitro selection,” selecting RNA
ligands against various organic dyes. The Szostak lab also coined the term
“aptamer.” Two years later, the Szostak lab and Gilead Sciences, employed
in vitro selection schemes to evolve single-stranded DNA ligands for
organic dyes and human coagulant, thrombin. From that time, the
researchers started to employ DNA aptamers more favorably than RNA
aptamers, given the intrinsic greater chemical stability of DNA in com-
parison with RNA.
SELEX consists of basic steps including incubation of targets with
oligonucleotide library (typically 1013–1016 random sequences), isolation
of the oligonucleotide — target complexes from unbound sequences and
finally amplification of the bound sequences by PCR in order to obtain an
enriched pool for the next round of selection (see Figure 2.9). By replicat-
ing the selection process, it is possible to enrich the bound sequences.
Throughout the entire process, the most critical step is the separation
by

of the bound sequences from unbound sequences, especially for SELEX

using purified targets. Thus many modified SELEX strategies have been
proposed to simplify this step or to enhance the efficiency of separation
[66]. The selected DNA or RNA sequences are finally cloned into bacteria
and sequenced with the aim to obtain a pool of optimal individual aptamer
candidates, which are finally labeled with reporters.
Aptamers are known also as nucleic acid antibodies and have
unique features, such as ease of chemical synthesis, excellent chemical
stability in physiological conditions, low molecular weight, lack of

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50 W. Pfeifer et al.

n target
tio
iliza
m ob
im
random
DNA library bead
y.

wash

SELEX PCR
amplification

unbound DNA

isolate bound DNA

DNA-target complex

Figure 2.9.  Scheme of systematic evolution of ligands by exponential enrichment

(SELEX) process for the synthesis of an aptamer for a protein target. Reproduced with
kind permission from Ref. [67].

immunogenicity, and ease of modification in comparison with protein

counterparts. They have the ability to bind to different targets such as
metal ions, small molecules, proteins, whole live cells, viruses and bacte-
ria with high affinity and specificity. Such features make aptamers very
promising candidates as molecular probes for novel class of electrochemi-
cal biosensors, as active elements for the recognition of extracellular
by

matrix signatures of cancer cells and target-specific ligands for therapeu-

tic purposes.
Given the possibility to engineer nucleic acid bases, a large number of
aptamer-based sensing systems have been developed for the efficient
detection of a wide range of molecules. Just to make some examples,
Plaxco et al. designed a series of aptamer-based electrochemical sensors
to detect ATP, cocaine and so on [68]; Zhao et al. designed a fluorescent
biosensor to detect proteins [69]; Ricci et al. designed allosterically con-
trollable, metal–ion triggered molecular switches to specifically recognize

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DNA as Building Material at the Nanoscale 51

mercury(II) or silver(I) ions [70]. Remarkably, aptamers can be generated

also to recognize target cells, including those expressing specific proteins
of interest. In this case, the selection process is defined cell-SELEX [69].
Typically, these aptamers are selected against live or — less commonly —
fixed cells. Partitioning between unbound sequences and cell-bound
sequences is carried out by centrifugation or washing. Remarkably, at
least one type of control cell has to be used for counter-selection in order
y.

to get rid of sequences that bind to proteins present at surface of both cell
types.
One of the major issues of initial SELEX technique is its time-­
consuming, labor-intensive nature. This problem was solved by the lab
of C. Cox that was able to automatize in vitro selection process, so
reducing the duration of the selection to some days [71, 72]. In addition,
another significative issue of DNA aptamers is that most of the reported
synthesized aptamers are selected and applied for in vitro studies,
whereas the in vivo stability of these aptamers is still not perfectly
known at present. Unmodified nucleic acid probes are susceptible to
nuclease digestion, so they are unstable in both intercellular and intra-
cellular environments. Therefore, innovation in SELEX protocols will
take into consideration aptamers with higher resistance under clinical
applications [66].

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DNA origami with layered crossovers. Angew. Chem. Int. Ed. Engl. 55(41),
12832–12835.
62. Sommerville, D.M.Y. (1929). Introduction to the Geometry of N Dimensions.
E.P. Dutton. New York, E.P. Dutton and company.
63. Edmonds, J., and Johnson, E.L. (1973). Matching, Euler tours and the
Chinese postman. Math. Program. 5, 88–124.
64. Ellington, A.D., and Szostak, J.W. (1990). In vitro selection of RNA mole-
y.

cules that bind specific ligands. Nature. 346(6287), 818–822.

65. Tuerk, C., and Gold, L. (1990). Systematic evolution of ligands by exponen-
tial enrichment: RNA ligands to bacteriophage T4 DNA polymerase.
Science. 249(4968), 505–510.
66. Lyu, Y., Chen, G., Shangguan, D., Zhang, L., Wan, S., Wu, Y., Zhang, H.,
Duan, L., Liu, C., You, M., Wang, J., and Tan, W. (2016). Generating cell
targeting aptamers for nanotheranostics using Cell-SELEX. Theranostics.
6(9), 1440–1452.
67. Meng, H.M., Liu, H., Kuai, H., Peng, R., Mo, L., and Zhang, X.B. (2016).
Aptamer-integrated DNA nanostructures for biosensing, bioimaging and
cancer therapy. Chem. Soc. Rev. 45(9), 2583–2602.
68. Lubin, A.A., and Plaxco, K.W. (2010). Folding-based electrochemical bio-
sensors: the case for responsive nucleic acid architectures. Acc. Chem. Res.
43(4), 496–505.
69. Huang, Y., Chen, J., Zhao, S., Shi, M., Chen, Z.F., and Liang, H. (2013).
Label-free colorimetric aptasensor based on nicking enzyme assisted signal
amplification and DNAzyme amplification for highly sensitive detection of
protein. Anal. Chem. 85(9), 4423–4430.
70. Porchetta, A., Vallee-Belisle, A., Plaxco, K.W., and Ricci, F. (2013).
Allosterically tunable, DNA-based switches triggered by heavy metals.
J. Am. Chem. Soc. 135(36), 13238–13241.
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71. Cox, J.C., and Ellington, A.D. (2001). Automated selection of anti-protein
aptamers. Bioorg. Med. Chem. 9(10), 2525–2531.
72. Cox, J.C., Rajendran, M., Riedel, T., Davidson, E.A., Sooter, L.J., Bayer,
T.S., Schmitz-Brown, M., and Ellington, A.D. (2002). Automated acquisition
of aptamer sequences. Comb. Chem. High. T. Scr. 5(4), 289–299.

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Chapter 3
DNA Sensors for the Detection
of Biomolecules and Biochemical
Conditions

Birgitta R. Knudsen*, Anni H. Andersen*,

Magnus Stougaard†, Giuseppe Arrabito‡, Raffaella Suriano§
and Yi-Ping Ho¶,||

* Department of Molecular Biology and Genetics

Aarhus University, Denmark
†
 Department of Clinical Medicine
Aarhus University, Denmark
‡
 Department of Physics and Chemistry
University of Palermo, Viale delle Scienze, Parco d’Orleans II
90128 Palermo, Italy
§
 Department of Chemistry, Materials and Chemical Engineering
“Giulio Natta”, Politecnico di Milano, Milano, Italy
¶
 Department of Biomedical Engineering
The Chinese University of Hong Kong
Hong Kong SAR, China
||
 ypho@cuhk.edu.hk

57

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58 B. R. Knudsen et al.

This chapter is a concise description of the outstanding applications of

DNA nanotechnology in the field of Biosensors. Strategies for top-down
fabrication of DNA sensors are described and signal transduction mecha-
nisms for sensors are reported.

3.1 Introduction
The general purpose of biosensors is to characterize specific properties of
a biological analyte or to characterize an interaction between analytes.
Biological affinity to the analyte is typically generated by the molecular
probe. This chapter focuses on the use of DNA molecules as probes. Once
an interaction between the DNA probe and analyte has occurred, the inter-
action is transduced into a detectable signal. Depending on the assay for-
mats and signal transduction methods, separation of bound and unbound
probes may be required. Typically, heterogeneous assays refer to assays
that require separation of bound and unbound species while homogeneous
assays do not. Separation can be accomplished through stringency washes
to rinse away unbound species provided the probes or analytes are
anchored to a suitable support.
Sensitivity, selectivity and sample volume are perhaps the most
widely touted measures for the performance of DNA sensors. The defini-
tions of sensitivity and selectivity may vary depending on the context of
assays. Generally speaking, sensitivity is defined as the lowest concentra-
tion or the minimal amount of analyte that can be detected against the
baseline of background. Selectivity refers to the ability of an assay to
distinguish between a specific analyte and the background or unspecific
analytes. Therefore, selectivity is typically quantified by the “signal-to-
noise ratio” (SNR), which is defined as a ratio between the signal pro-
duced by the targeted analytes and the noise generated by the interfering
species. The sample volume is straightforward and defines the minimal
volume of sample required for a particular assay.
Based on the timescales during which measurements are recorded, the
assays can also be categorized into equilibrium or real-time assays. In
equilibrium assays, the measurements are taken after the steady state is
reached. For example, heterogeneous assays require stringency washes.
Therefore, the measurement is normally taken when the assay reaches a

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 59

steady-state equilibrium between probe-analyte binding. For homogene-

ous assays, provided a proper signal transduction technique, measure-
ments can be recorded real-time as the reaction is ongoing to acquire
kinetic information of the reaction.
As an overview to the audience interested in utilizing DNA sensors
for various purposes, this chapter aims to present the state-of-art fabrica-
tion of DNA sensors, commonly used signal transduction mechanisms,
and different sensing strategies for the detection of nucleic acids, proteins,
enzymes and their activities as well as biochemical conditions and small
molecules.

3.2  Construction of DNA Sensors

To specifically assay the interactions between the DNA probe and the
analytes, it is necessary to immobilize DNA onto a detectable compo-
nent, such as a solid surface or the surface of nanoparticles, forming a
DNA sensor without interfering with the base pairing. A variety of
methods have been used over the last decade to fabricate and develop
miniaturized DNA sensors, given the large breadth of applications
found for these devices. The fabrication methods can be generally
divided into two major categories: “bottom-up” and “top-down” meth-
ods according to the processes involved in creating DNA ordered struc-
tures on substrates. A bottom-up approach uses molecular or atomic
components as building blocks to build up multi-level structures and
self-organizing assemblies based on intermolecular and supramolecular
interactions and complex mechanisms [1]. From the bottom-up perspec-
tive, the immobilization of DNA is typically achieved through either
adsorption or covalent binding, similar to those for the enzyme based
biosensors [2, 3]. Adsorption is the simplest as it does not require modi-
fications of the nucleic acids. However, the specificity may be of a
concern. On the other hand, covalent conjugation is more specific as
there are particular sites that can be modified on the nucleic acids,
namely the available bases, the sugar or the phosphate groups to pro-
duce derivatives for incorporation of various functional groups such as
–SH, –NH2, –COOH, or –biotin to covalently attach with the to-be-
conjugated molecules. The conjugation chemistry and protocols for the

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60 B. R. Knudsen et al.

modification of nucleic acids have been established and reviewed else-

where [4] and thus are not elaborated herein.

3.2.1  Top-down fabrication of DNA sensors

As opposed to the bottom-up approach, top-down approaches correspond
to the use of fabrication tools controlled by external experimental param-
eters to create DNA patterns and functional devices with the desired
shapes and characteristics [5]. The ability to ensure a controllable place-
ment of the DNA molecules on desired regions and potentially on large
areas makes top-down methods the most widespread approaches for the
fabrication of DNA sensors. Among the top-down approaches, various
lithography-based techniques have been employed for patterning 2D DNA
features.
One established top-down approach is dip-pen nanolithography (DPN),
which involves the selective deposition of molecules onto a surface in a
regular arrangement of dots or shapes by means of atomic force micros-
copy (AFM). More specifically, the DNA patterning using the DPN tech-
nique involves the use of one or several tips, which are previously dipped
in a solution of the molecules to be deposited, commonly referred to as
the “ink.” The ink is transferred to the substrate when the AFM tip is very
close to the substrate (few nanometers). This close contact with the sur-
face enables the AFM tip to release the material of interest according to a
predefined pattern obtained with the movement of scanning tips. This
method offers the possibility to pattern features with a lateral resolution of
around 50 nm, allowing the fabrication of high-resolution DNA nanoar-
rays [6]. Among the numerous strategies developed for DNA patterning
by means of DPN, one of the most employed methods is the direct-write
deposition of oligonucleotides on the substrate. This was demonstrated by
Demers et al. to generate covalently bonded patterns of oligonucleotides
on gold and SiOx substrates [7]. This work also was the first example
of functionalization of AFM tips to achieve a successful deposition of
DNA. It could be achieved because the surface of silicon nitride tips was
modified with a chemical compound, i.e. 3-aminopropyltrimethoxysi-
lane, to promote a good adhesion of DNA ink molecules to the tip sur-
face. By using these modified tips, functionalized oligonucleotides can

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 61

be patterned on different surfaces with a high-quality resolution, preserv-

ing their sequence-specific activity as verified by hybridization with com-
plementary probes. The use of a carrier such as an agarose hydrogel has
also been proved to have many advantages over the direct deposition of
oligonucleotides in a DPN writing process, resulting in a fast and efficient
patterning of oligonucleotide nanostructures, which maintain their bio-
logical activity and specificity (Figures 3.1(a)–3.1(c)) [6].
DPN can also be used in the so-called “indirect” approach to obtain a
certain pattern, which then directs the immobilization of biomolecules
from the solution to the surface. The biomolecule indirect deposition
approach requires the ability to anchor these molecules to the substrate
through specific interactions, which minimize non-specific surface inter-
actions. For example, the DNA molecules can be immobilized in an
ordered arrangement via electrostatic interactions between positively
charged amino-terminated arrays printed by DPN and a solution of nega-
tively charged oligonucleotides [8]. Alternatively, immobilization can be
achieved via covalent bonds by coupling an amino-functionalized DNA
molecules to a DPN-printed pattern with carboxylic acid terminal groups
by the formation of an amide bond [9]. Although the DPN technique
offers the freedom of printing any kind of patterns and high resolutions
with a minimum feature size of 15 nm (for 16-mercaptohexadecanoic acid
on gold), the process is serial and therefore is slower than the parallel
techniques. To overcome the limitation of printing speed, multiple probes
and tip arrays have been developed, thus allowing a large-volume produc-
tion and high throughput [10, 11].
Another lithographic technique based on scanning probe microscopy
is the nanografting method. When compared to other top-down fabrication
techniques, the nanografting process shows greater control over the size,
geometry and position of biomolecules on the surface. Usually, the nano-
grafting method is employed on surfaces previously modified with self-
assembled monolayers (SAMs). The process can be divided in two steps:
(a) a pre-existing SAM is removed via nanoshaving using an AFM tip as
a razor in the presence of surfactant molecules, having a greater affinity
with the substrate than those removed by the tip; (b) once the previous
SAM is removed from the addressable area, another different SAM can be
obtained over the shaved surface.

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62

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B. R. Knudsen et al.
Figure 3.1.    (a) Schematic representation of ink and matrix components; (b) an illustration showing the process of agarose-assisted
DPN; (c) epifluorescent microscope image of a 15 × 20 array of 500 nm Cy3 labeled oligonucleotide features generated in parallel
from a 12-tip cantilever array. Adapted with permission from Ref. [6]. Copyright © (2016) American Chemical Society.
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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 63

For a successful deposition by nanografting, SAMs have to meet

some requirements: (i) the pre-existing SAM must be easily removable by
the AFM tip; and (ii) the successive SAM has to be formed very quickly.
For these reasons, thiol-gold combination is usually employed for the
nanografting, particularly due to the rapid formation of homogeneous
monolayers of thiols on gold surfaces. Liu et al. demonstrated the deposi-
tion of oligonucleotides functionalized with a thiol group by direct nano-
grafting. Due to the presence of pre-existing SAM, it is possible to drive
the adsorption of DNA molecules and also to prevent lateral diffusion of
DNA, resulting in an excellent feature control [12]. Another advantage of
this direct nanografting method is a 3D arrangement of oligonucleotides,
which are almost perpendicular to the surface and placed in thin lines
wide up to 20 nm surrounded by the pre-existing SAM, thus enabling an
easier subsequent hybridization.
Moreover, electron beam lithography (EBL) can also be used for
depositing DNA arrays, as shown by Zhang et al. [13]. An advantage of
this lithographic technique is its compatibility with the normal techniques
of microfabrication developed in the electronics field, thus allowing a
simple integration of biomolecules in electronic sensors or Bio Nano
Electro-Mechanical System (bioNEMS). However, EBL only operates in
an atmosphere of ultra-high vacuum, strongly limiting the potential of this
technique in manufacturing multi-component nanoarrays.
Another lithographic technique is microcontact printing (μCP),
widely used for the deposition of organic molecules and biomolecules on
large areas (> cm2). The μCP process, developed by Kumar and Whitesides
[14], usually employs an elastomeric mold. The fabrication of the mold
starts from the creation of a negative master mold, on which a liquid pre-
polymer is poured. Once the polymerization of the prepolymer is obtained
through a heating treatment (curing), the mold is separated from the mas-
ter and ready for use. The mold is then dipped in the solution containing
the molecules to be deposited on the surface. After a sufficiently long
incubation time which depends on the ink (1–45 min), the mold is dried
and pressed on the substrate. Due to the conforming contact between the
substrate and the relief parts of the mold, the molecules are transferred to
the surface. Once the master is available, the mold fabrication can be
completed within a couple of hours. Polydimethylsiloxane (PDMS) is the
material of choice for the preparation of the mold, because its low-cost

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64 B. R. Knudsen et al.

and suitability for normal laboratories. The entire deposition process can
also be performed without the need of a clean room. All these factors
make this technology very flexible, fast and affordable for most laborato-
ries, contributing to its popularity. Nyamjav and Holz demonstrated the
use of μCP as a robust, reliable and inexpensive method to produce prede-
signed, spatially controlled, high density microarrays of DNA molecules
directly on silicon oxide substrates. The coupling of oligonucleotides to a
silicon substrate was achieved by silanizing an acrylamide-terminated
DNA, resulting in arrayed oligonucleotides with a retained biological
function [15].
With the aim of manufacturing low-cost electronic biosensor devices,
one of the most promising technologies is inkjet printing. A large versa-
tility is shown by this patterning method, which is also suitable for pro-
totyping. This approach is based on the ejection of a liquid ink through
micrometer-sized nozzles under a pressure pulse. It enables the deposi-
tion of very small volumes of ink (from pL to nL) in a rapid procedure,
achieving high pattern precision, micron-sized resolution and a good
reproducibility [16].

3.3  Signal Transduction Mechanisms

The signal transduction for DNA sensors may be achieved through opti-
cal, electrical, mechanical or magnetic routes. Optical detection is perhaps
the most commonly used method due to the high sensitivity of optical
systems and due to the legacy from assays developed with molecular fluo-
rophores and dyes. Optical detection methods are typically based on fluo-
rescence emission, surface plasmon resonance (SPR), scattering,
colorimetric change, Förster resonance energy transfer (FRET) or surface
enhanced Raman scattering (SERS). This section will review the most
widely adopted signal transduction mechanisms based on fluorescence
and plasmonics.

3.3.1  Fluorescence-based sensing

Fluorophore conjugates, which provide fluorescence or act by changing
the spectral properties of the sample, are the basis for a wide range of

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 65

DNA sensors. For example, premier Cyanine Dyes (e.g. PicoGreen,

SYBR Green) and cyanine dimers or monomers (e.g. TOTO or YOYO
family), intercalate DNA to hide their hydrophobic chains from the sur-
rounding aqueous solution. This results in increased fluorescent output by
several orders of magnitude [17–19] and has been utilized extensively in
genomics research. Aside from the conventional organic fluorophores, the
utility of nanoparticles, namely colloidal particles of 5–50 nm in diameter,
such as semiconductor nanocrystals and metallic nanoparticles, have fun-
damentally changed the bioanalytical measurement landscape [20–22].
Luminescent semiconductor nanocrystals, colloquially known as quantum
dots (QDs), stand amongst the most exciting research tools in chemistry,
physics and biology. These inorganic fluorophore crystals, normally com-
posed of periodic groups of II–IV (e.g. CdSe) or III–V (e.g. InP) material,
have demonstrated superior optical properties, such as narrow, symmetric
and precise size-tunable emission spectra and broad excitation spectrum.
Other commonly discussed benefits of QDs in comparison to organic
fluorophores include enhanced fluorescence stability and negligible pho-
tobleaching [23]. Consequently, much attention has been focused on the
utilization of QD for the construction of DNA sensors [24, 25], particu-
larly for those aiming for multiplexed detection [26, 27].

3.3.1.1  Förster resonance energy transfer

FRET is a commonly used strategy in DNA sensors. FRET occurs when
the energy of the donor is non-radiatively transferred to a nearby acceptor
molecule via a dipole–dipole interaction, leading to a decrease in the donor
emission and an increase in the acceptor emission, in such a way that the
excited state lifetime of the donor is diminished [28, 29]. The energy trans-
fer efficiency, E, is a function of donor–acceptor separation r, serving as
an important measure to the energy transfer process. Equation (1) presents
the general definition describing the fraction of excitations transferred
from the donor to the acceptor non-radiatively, where kT is the rate of non-
radiative energy transfer and tD is the excited-state radiative lifetime.

kT R06
E= = (1)
t D−1 + kT R06 + r 6

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66 B. R. Knudsen et al.

The Förster radius R0 refers to the distance at which the energy trans-
fer efficiency is 50%. This is typically used to characterize the FRET
relationship of a particular donor–acceptor pair. For most FRET pairs, R0
is typically a few nanometers, enabling optical measurements of changes
in donor–acceptor distance with angstroms resolution, leading to a
“nanoruler” probing intramolecular or intermolecular distance.
Ideal FRET occurs when there is appreciable spectral overlap between
the emission spectrum of the donor and the absorption spectrum of the
acceptor, while crosstalk caused by spectral overlap, of the donor and
acceptor emission is minimum. With this request in mind, organic fluoro-
phore FRET pairs often suffer from direct acceptor excitation (excitation
of the energy acceptor at the meantime of exciting the energy donor) and
crosstalk (spectrum overlap between donor and acceptor emissions), due
to their broad adsorption and emission spectra. To this end, there is an
emerging effort on involving QDs in the FRET process, since this over-
come some of the limitations associated with conventional organic FRET
pairs [25, 30–32].

3.3.2  Plasmonics-based sensing

Plasmonics is the study of the interaction between an electromagnetic
field and free electrons in a noble metal, such as gold or silver [33]. The
unique properties of plasmonic materials derive from the plasmon oscilla-
tion, i.e. the collective motion of conduction band electrons relative to
fixed positive ions. The design of artificial plasmonic structures has
shown enormous importance for the production of biosensors, for instance
in the form of SPR [34]. SPR can be defined as the phenomenon, which
occurs when polarized light, under conditions of total internal reflection,
strikes a conducting gold layer at the interface between media of different
refractive index such as the glass of a sensor surface (high refractive
index) and a buffer (low refractive index). The resonance condition is
established if the frequency of incident photons matches the natural fre-
quency of surface electrons of a conducting gold layer oscillating against
the restoring force of positive nuclei.
Remarkably, by tuning geometries and order in nanomaterials, it is
possible to obtain plasmonic effects showing phenomena such as absorb-
ance and scattering. Such unique features make these materials a hot

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 67

research topic due to applications such as signal enhancement for bioim-

aging [35] and biosensing [34]. The commonly used approach to build up
nanomaterials with desired plasmonic properties is by conventional
top-down photolithography. However, these approaches suffer from
high costs and are not suitable to construct 3D structures. In this regard,
DNA nanotechnology allows for assembling nanomaterials by bottom-
up approaches in order to build up plasmonic metallic nanostructures
showing high complexity and hierarchy. In fact, the versatile chemistry
of DNA allows fine-tuning of the optical properties of plasmonic nano-
structures, such as coupling nanoparticles surface plasmons in chiral
assemblies.
For instance, Kuzyk et al. [36] designed DNA origami for producing
plasmonic structures containing nanoparticles arranged in left- and right-
handed nanoscale helices. They employed DNA origami 24-helix bundles
having nine helically arranged attachment sites for plasmonic particles
coated with single stranded DNA, i.e. gold nanoparticles with a diameter
of 10 nm or 16 nm. By bulk measurements, such structures are found to
exhibit defined circular dichroism and optical rotatory dispersion effects,
which are due to the collective plasmon–plasmon interactions of the
nanoparticles positioned.
As an example of an active plasmonic system, Zhou et al. demon-
strated the possibility to fabricate a plasmonic nanorod that can execute
directional, progressive and reverse nanoscale walking on 2D- or 3D DNA
origami. The designed walker has an anisotropic gold nanorod and DNA
strands as feet that allow it to move on a DNA-origami [37]. More
recently, Gur et al. fabricated a new class of plasmonic waveguides by
thoroughly investigating the effect of several parameters that can influ-
ence the attachment yield of oligonucleotide-functionalized AuNPs to
six-helix bundle (6-HB) DNA origamis to produce self-assembled plas-
monic waveguide precursors. They found out that high ionic strength
(300 mM NaCl and 12 mM MgCl2) is necessary to stably hybridize the
AuNPs to the tethers on the DNA origami and that hybridization of
AuNPs to 6-HBs takes seconds, and aggregate formation is irreversible [38].
Copp et al. investigated the effects of dielectric environment and cluster
shape on electronic excitations of fluorescent DNA-stabilized silver
clusters. Their results suggested the possibility to analyze dielectric
environments at length scales of around 1 nm [39].

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68 B. R. Knudsen et al.

On the other hand, the plasmonic properties of dimeric nanostructures

are also reported. For example, Acuna et al. [40] designed a DNA-origami
pillar having docking sites to allow for the assembly of nanoparticle
dimers apart from each other in a distance of 23 nm. In this manner, self-
assembled nanoantenna that allowed for enhanced fluorescence intensity
in a plasmonic hotspot of zeptoliter volume was created. They carried out
direct visualization of the binding and dissociation of short ATTO
655-labeled DNA strands, as well as the conformational dynamics of a
DNA Holliday junction in the hotspot of the nanoantenna, thus showing
the compatibility with single-molecule bioassays.
As an alternative to fluorescence detection, some reports deal with
Raman detection, a technique which is based on inelastic scattering
of monochromatic light, usually from a laser. The laser light interacts
with molecular vibrations, phonons or other excitations in the system,
resulting in the energy of the laser photons being shifted up or down.
Such energy provides useful information about the vibrational modes
in the system. Thacker et al. [41] carried out plasmonic coupling
between two 40 nm gold nanoparticles held with a gap of about 3.3 nm
by a DNA origami suitable for Raman detection. They employed such
dimers to measure Rhodamine 6G, a model Raman analyte, demon-
strating that only in the presence of gold nanoparticle dimers, it was
possible to acquire Raman spectra. They also obtained SERS spectra of
nanoparticles coated with ssDNA for sequence-­specific DNA detection
showing an average enhancement of 4–5 orders of magnitude in the
scattering cross-section in comparison with bulk measurements — see
Figure 3.2(a).
Finally, in a very recent report, Sinha et al. [42] showed the possibility
to fabricate DNA-mediated gold nanoprism based optical antennas, which
can enhance two-photons-imaging capability in the 1100 nm biological II
window. In the process of two-photon fluorescence (TPF), nanoparticles
or organic molecules absorb two low energy photons and emit a single
excited photon at an energy level higher than that of the absorbed photons.
The report demonstrated enhanced second harmonic generation (SHG)
and TPF properties by several orders of magnitude via DNA-driven
­plasmon-coupled organization into gold nanoprism assembly structures
using human Hep G2 liver cancer cells as a model.

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 69

Baseline

ttt ttt ttt ttt ttt ttt t ttc ctc tac cac cta cat
SEQUENCE 1 SEQUENCE 2 dsDNA level
Threshold
Intensity (counts/(mW s)) Intensity (counts/(mW s))

600 800 1,000 1,200 1,400 1,600 1,800

SEQUENCE 1 100pA Searching window
1,000 1,084 1,576 1ms
400

Incubation Translocation
200

C1
20% SEQUENCE 1 1,260 1,485
80% SEQUENCE 2
1,200

C2
800 736

400 C3
100pA
600 800 1,000 1,200 1,400 1,600 1,800
1ms
Wavenumber shift (cm–1)
(a) (b)

Figure 3.2.    (a) DNA origami based surface-enhanced Raman scattering for DNA sens-
ing. Gold nanoparticles — plated on a gold coated silicon surface — are coated with two
different types of DNA sequences (sequence 1 is made of 19 thymines and sequence 2
adenine and cytosine in an 80:20 ratio). SERS spectra can distinguish the two different
analytes, since SERS spectra of sequence 1 gives peaks corresponding to the DNA
­backbone (1000 cm−1) and thymine (1084 cm−1), whereas SERS spectra of sequence 2
shows peaks corresponding to adenine (736, 1260 and 1485 cm−1) and cytosine (1260 and
1485 cm−1). Reprinted with permission from Macmillan Publishers Ltd: Nature
Communications (Ref. [41]), copyright © (2014). (b) At the left, representation of DNA
carrier (black line) and protein complexes (black line with cuboid attached) translocating
through a nanopore driven by the electric field. At the right, carrier translocation event
showing an extra second level current drop in the middle of the event, due to the targeted
protein binding. By decreasing protein/carrier concentration ratio (c3 < c2 < c1), less occu-
pied events are found. Reproduced from Ref. [47]. Copyright © (2016) American
Chemical Society under Creative Commons Attribution (CC-BY) License.

3.3.3  DNA nanopore-based sensing

DNA nanopore-based sensing represents one of the most interesting appli-
cations of DNA-based nanostructures. Under an application of constant
voltage, nanopore-based detection measures the ionic current between
two electrolyte solutions separating by a single nanoscale pore. As

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individual molecules translocate the pore, the flow of ions passing through
the pore reduces and therefore the measured current [43]. The first exam-
ple of nanopore-based sensing was presented by Bayley and coworkers
with the α-hemolysin protein [44], a nanopore from bacteria that causes
lysis of red blood cells. The report showed the capability of nanopore-
based sensing for DNA sequencing, since all four bases can be identified
by the measured ionic current. Furthermore, research efforts have permit-
ted the understanding of how DNA molecules interact with nanopore
system during the translocation process. When bound with analytes
through covalent/non-covalent interactions, DNA hybrids produce charac-
teristic current signals when translocated through nanopore which permit
analytes identification. Researchers have also employed DNA carriers
molecules, long DNA strands which have specific binding sites for target
analytes in solution. Such DNA constructs are able to interact with analyte
(typically a protein) and, upon translocation through the nanopore, spe-
cific current signals of the DNA–analyte complex are measured. Analyte
sensing can be carried out via signature current events generated by dif-
ferent DNA–analyte interactions, such as DNA hybridization, DNA
aptamer–analyte binding, DNA metal ion binding, DNA protein interac-
tions and host–guest interactions [43].
In addition to α-hemolysin protein, synthetic DNA-origami nano-
structures have also been applied as a nanopore by two different
approaches: (1) by trapping a DNA-origami structure at the mouth of a
solid state nanopore, and (2) by an insertion of a DNA-origami structure,
coated with hydrophobic moieties, into a lipid bilayer.
As an example of the first approach (i.e. inserting DNA nanostruc-
tures inside solid state nanopore), Bell et al. reported a strategy to repeat-
edly insert and eject funnel shaped DNA origami designed with a
constriction of 3 × 3 double-helix widths (7.5 nm × 7.5 nm) through solid-
state nanopores with diameters around 15 nm for detecting model DNA
analytes, such as λ-DNA molecules [45]. Hernández-Ainsa et al. com-
bined DNA-origami structures with glass nanocapillaries to reversibly
form hybrid DNA-origami nanopores [46]. As a very recent example,
Kong et al. [47] combined solid-state nanopores and DNA carriers to
quantify nanomolar level of protein concentration. They could determine
protein concentration by measuring the fraction of DNA carriers which

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showed a secondary signal indicating the presence of a protein. They used

two model systems, namely biotin–streptavidin and digoxigenin−antidi-
goxigenin — see Figure 3.2(b). The working protein concentrations were
at nanomolar range, and microfluidic chips with ~10 μL volume were
used, so the total amount of protein needed was only 10 fmol.
The second approach is conducted by the insertion of a structure into
a lipid bilayer. In this scenario, it is possible to mimic membrane proteins
which insert into cell membranes for controlling the transport of mole-
cules and ions. The charged phosphate backbone of DNA is hydrophilic
and so the DNA nanostructure must be decorated with hydrophobic
chemical groups to overcome the large free energy barrier to form a hole
in the lipid bilayer. In a fundamental report, Langecker et al. linked cho-
lesterol tagged oligonucleotides to a stem and barrel DNA-origami struc-
ture which formed a pore 2 nm wide and 42 nm in length. Such
nanostructured pores were capable of insertion inside lipid vesicles with
the desired orientation [48].

3.4  Detection of Nucleic Acids

DNA sensors for detection of nucleic acids usually rely on the specific
Watson–Crick base-pairing between polynucleotides. Some of the first
were simply labeled oligonucleotides which could be hybridized to a tar-
get sequence. Although these were robust they had low resolution and
were incapable of detecting minor stretches or variations in nucleic acids.
Specific detection of small sequence stretches or even single nucleotide
variations required highly specific DNA sensors in combination with
powerful amplification strategies. One of the first examples of such highly
specific DNA sensors was the so-called Padlock probes [49]. Padlock
probes are DNA oligonucleotides consisting of two target-complementary
DNA sequences, linked by a freely designable DNA sequence typically
used for probe identification. Upon hybridization to the target sequence
the 5′- and 3′-ends of the padlock probe are brought into proximity allow-
ing the open circular DNA structure to be converted to a closed circular
DNA structure by the action of a DNA ligase [49]. Due to the helical
structure of DNA, padlock probes are topologically linked to the target
DNA after hybridization and ligation. Padlock probes, therefore, allow

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stringent washing conditions and thus reduced background staining

despite a short hybridization sequence [50, 51]. This, combined with the
specificity provided by the ligation event, which is sensitive enough to
distinguish single base differences [52] enabled detection of single nucle-
otide variations in the alpha satellite of centromeres in human chromo-
somes [53].
The significant advantage of padlock probes is due to their capability
to change from an open circular DNA structure to a closed circular DNA
structure. This closed DNA structure can be used as template for subse-
quent amplification strategies such as Rolling Circle Amplification (RCA)
[54–58], or Polymerase Chain Reaction (PCR) [59, 60]. To better allow
multiplexing, the strategy of padlock probes was further modified in what
was named Molecular Inversion Probes (MIP) [59] and Connector
Inversion Probes (CIP) [61]. Both types of probes closely resemble pad-
lock probes but are designed to hybridize with either a single nucleotide
gap (MIP) or a larger gap (CIP) between the 5′- and 3′-end. Upon hybridi-
zation, the gap is filled by a DNA polymerase upon addition of either the
specific nucleotide complementary to the single nucleotide gap (for the
MIP) or all four nucleotides (for the CIP). The readout was performed
using genotyping arrays with sequences complementary to the linker
sequence or using sequencing [59, 61, 62].
Detection of RNA targets using padlock probes in combination with
RCA [55, 63, 64] or hyperbranched RCA (hRCA) [65–67] have also been
reported. However, although DNA ligases can ligate DNA using an RNA
template it is less efficient than using a DNA based template [68–72].
Therefore, different solutions have been proposed to circumvent the use
of RNA as a template for the ligation. One solution was to use preligated
closed circles primed from the RNA-target [67, 73, 74]. A second method
was to use a DNA sensor termed turtle probe designed to form a second-
ary hairpin structure bringing the 5′- and 3′-end into proximity facilitating
ligation to form a closed circle which could serve as template during RCA
primed from the target RNA [75, 76]. The last technique was to reverse
transcribe the RNA to cDNA using an LNA primer, degrade the RNA
to make a single stranded target, and detect the cDNA with padlock
probes [77].

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A different group of DNA sensors for detection of RNA are based

on DNAzymes, also called deoxyribozymes, which are DNA sequences
with catalytic functions. Unlike RNAzymes, also referred to as ham-
merhead ribozymes, which have been found widely distributed in the
nature [78], ranging from viroids and viral satellites [79, 80] to humans
[81], no naturally occurring DNAzymes have been reported. DNAzymes
are a result of in vitro selection and the first reported catalyzed Pb2+-
dependent cleavage of RNA [82]. Soon after several others were reported
[83, 84], including the general purpose RNA cleaving 10–23 and 8–17
DNAzymes [85] and the horseradish peroxidase (HRP) mimicking
DNAzyme [86, 87]. DNAzymes have formed the basis for many differ-
ent sensors using different read-outs e.g. electrochemical [88, 89], fluo-
rescent [90], or colorimetric [91].

3.5  Detection of Proteins

The development of synthetic DNA/RNA aptamers via the Systematic
Evolution of Ligands by Exponential Enrichment (SELEX) technology
[92–94] have led to the development of aptamers capable of recognizing
specific proteins [94]. The development of aptamers have illustrated that
protein detection can be performed using DNA sensors in assays resem-
bling antibody sensors without the need for antibody development.
Moreover, as described for nucleic acid detecting sensors, the use of DNA
sensors for protein detection allow the use of a variety of readouts e.g.
direct labeling of the aptamer with fluorescence or biotin followed by
direct or indirect visualization using streptavidin-conjugated Phycoerythrin
(PE) or QD [95]. Alternatively, different amplification strategies have
been used such as RCA primed directly from the aptamer [96–101] or
PCR of the aptamer [102, 103].
DNA sensors in the form of aptamers have been used for directly
sensing biomolecules in solution relying on proximity ligation [104] or
proximity extension [99]. Proximity ligation or extension is based on the
principle that two or more target binding sensors e.g. aptamers have to be
brought in proximity before an amplification (e.g. RCA or PCR) leading
to detectable signal can take place [104, 105]. Although DNA sensors in

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the form of aptamers seem appealing for proximity assays, the lack of
suitable aptamers has led to the use of DNA labelled antibodies rather than
the DNA aptamers [106, 107].
Another promising family of sensors for detection of proteins in solu-
tion is based on structure-switching aptamers. Several different types of
structure-switching aptamer based DNA sensors have been developed
using various strategies. One type changes into an exonuclease resistant
structure upon binding of the target protein thus preventing degradation of
the DNA aptamer and resulting in a fluorescent signal by addition of an
intercalating fluorescent dye [108]. Although beautiful in its simplicity,
this approach may be challenged by its specificity in the presence of DNA
binding proteins. A second nuclease based structure-switching aptamer
system changes from a hairpin shaped structure into a partly single-
stranded stretch of DNA upon target protein (thrombin) binding to an
aptamer. The single-stranded stretch of DNA can hybridize to a fluoro-
phore–quencher conjugated probe generating a double-stranded substrate
for a nicking endonuclease that can cleave the probe and thus causing an
increase in fluorescence through separation of the quencher and fluoro-
phore [109]. A different approach was demonstrated by Yang et al. who
developed a structure-switching aptamer that could be circularized only
upon specific binding of its protein target generating the template for a
subsequent RCA [110]. Lastly, a universal structure-switching aptamer
based system has been presented in which an aptamer and a padlock probe
compete for binding to a specific oligonucleotide. Upon binding of the
target protein to the aptamer, the oligonucleotide is free and can serve as
a template for the padlock probe that can be amplified using RCA [111].
This system presents the significant advantage of being easily adaptable
to the detection of other biomolecules although the target binding
sequence has to be changed for each new target detected.

3.6  Detection of Enzymes

DNA sensors for detection of enzyme activity may find use in many dif-
ferent fields of basic or applied science for e.g. basic enzyme characteriza-
tions, drug screening, prognostics or diagnostics [112–114]. One of the
most straightforward advantages of DNA sensors tailored for measuring

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enzyme activity, is that they can easily be converted to “real time sensors”
allowing investigation of kinetic parameters and the acquisition of large
data sets from single reactions.
Examples of DNA based real time sensors for monitoring enzyme
activities are a large variety of DNA oligonucleotides coupled to quencher-
fluorophore or other FRET pairs designed in such a way that the two parts
are either brought together or separated upon reaction with the target
enzyme. In this manner interaction with the target enzyme results in either
gain or loss of signal depending on the specific design of the sensor.
Examples of the reporter fluorophores include both organic fluorophores
and semiconductor nanocrystals (e.g. QDs). Moreover, readout systems
based on carbon nanoparticles, or gold nanorods have also been reported.
The different sensor systems have been demonstrated for detection of dif-
ferent enzyme target activities including endonuclease induced DNA
cleavage [115–120] or binding (the latter by using single molecule FRET
[121]), DNA polymerases [122], DNA repair activities [123–126] and
various topoisomerases [127–130]. These types of sensors excel by the
ease and speed by which large data sets can be obtained, and for this rea-
son they have been demonstrated of particular use for kinetic analyses.
However, with regard to sensitivity they are inferior to more tedious pro-
tocols involving several amplification steps as e.g. RCA-based protocols
(see below).
Other examples of real time DNA sensors for monitoring enzyme
activities are the so-called label free sensors. In one setup, the detection in
the DNA sensor was transduced into electronic signals by a monolayer
graphene field-effect sensor containing DNA oligonucleotides coupled to
monolayer graphene strips that allow real-time measurement of human
topoisomerase I-mediated cleavage and ligation [131].
Already at this early stage of their development, real time sensors
have demonstrated their superiority when it comes to basic investigation
of enzymatic reactions including the effect of various inhibitors or poten-
tial small molecule drugs, and as such real-time sensors hold promise for
future drug screening programs [132]. Furthermore, the inclusion of real
time sensors in existing assays, such as ELISA have enabled simultane-
ously detection of protein amount and enzymatic activity in extract from
cells and tissue [133]. Several examples of using real time DNA sensors

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for measuring various enzyme activities in crude extract from biological

samples e.g. tissue or cells have also been reported [124, 126, 128, 130].
Such measurements may be of importance for diagnostic or prognostic
purposes of specific enzyme activities that may be used as biomarker for
disease and/or treatment response.
In cases where the available samples are limited or the enzymatic
marker of interest is rare, the optical or electrochemical signals gener-
ated directly by the enzymatic reaction with a real time DNA sensor may
be insufficient for detection. Hence, to obtain maximal detection levels
the sensor module may be combined with one or more signal amplifica-
tion steps. Sensors composed of DNA provides the benefit of being
compatible with a large number of different amplification methodolo-
gies including PCR [134], RCA [112], Loop Mediated Amplification
[135], Strand Displacement Amplification [136] and many more [137].
Of these, RCA has been used for signal enhancement in relation to the
analyses of various enzyme activities. The method presents the advan-
tage of being an isothermal reaction keeping an one-to-one stoichiome-
try between the RCA product and the original DNA molecule that is
amplified. Moreover, RCA generates a long tandem repeat product that
can be detected in bulk sample or at the single molecule level. Single
molecule visualization techniques include AFM [138–141], Transmission
Electron Microscopy (TEM) [142–144], and fluorescence microscopy
(following labelling of the RCA product with fluorescence probes or
nucleotides) [145–148]. Methods for bulk sample analyses include
colorimetric readout by employing enzyme or nucleic acid catalyzed
color formation e.g. oxidation of TMB (3,3′,5,5′-tetramethylbenzi-
dine), a chromogen that generates a blue color when oxidized. As
mentioned above, because RCA is performed using an isothermal pro-
tocol, the reaction follows simple linear kinetics. This is in contrast to
the exponential amplification of PCR and enables straightforward
quantitative measurements of enzyme activities without reference to
multiple standard samples [54, 145, 148, 149]. In order to allow
enzyme activity detection to be combined with signal amplification by
RCA we recently developed a family of oligonucleotide based sub-
strates that are circularized only upon reaction with their specific target

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 77

enzymes [145, 146, 148, 150]. By converting the DNA circle product of

each enzymatic reaction to a long tandem repeat RCA product this sys-
tem allowed direct analysis of endogenous enzyme activity at the single
catalytic event level. Taking advantage of a sensor oligonucleotide that
reacts only with one specific partner enzyme i.e. a specific topoisomer-
ase or recombinase [145, 146, 148] to generate a DNA circle that can
template RCA, the Rolling circle Enhanced Enzyme Activity Detection
(REEAD) setup is suitable for detecting enzyme activity in crude sam-
ples. Moreover, REEAD has proven robust, allowing specific and multi-
plexed detection of different enzymes in extracts from small samples of
human cells or, when combined with droplet microfluidics, even in sin-
gle cells (Figure 3.3) [151]. Since REEAD is not a real-time method it
is less well suited for kinetic analyses than the above described real-time
sensors. However, due to the multiple signal amplification steps incor-
porated in the REEAD the method is typically superior to the real time
sensors with respect to sensitivity showing a detection limit of 0.1 fmol
of purified enzyme [145]. In case high sensitivity is not needed a simpli-
fied but high throughput version of REEAD using real time measure-
ment of the RCA have been demonstrated [152].
Besides basic analyses of the target enzymes such as topoisomerases
or recombinases, the REEAD assay may be useful for analyzing the
molecular background behind development of disease or drug resistance
in e.g. cancer cell populations. Some of the enzymes that have been moni-
tored by REEAD are members of the DNA topoisomerase family of
which several are cellular targets of routinely used anti-cancer chemo-
therapeutics [153–155]. Many of these drugs act by converting the topoi-
somerase activity to a cell poison and, consequently, their anti-cancer
effect depends on the topoisomerase activity level in the cells. For this
reason, the topoisomerase specific REEAD assay was envisioned as a
powerful future cancer-prognostic tool suitable for analysis of small
biopsy samples [145]. More recently, the development of a REEAD setup
specific for detecting the activity of topoisomerase I expressed in the
malaria causing Plasmodium parasites [148, 156] opened up for the appli-
cation of REEAD in diagnosis of infectious diseases in human or life
stock. Combined with droplet microfluidics the multiple signal

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Figure 3.3.  Diagnosis by RCA based sensor systems. Upper panel (left) shows the
extraction unit composed of a droplet microfluidics chip. An oil phase is injected in an oil
inlet while water phases containing the sample to be analyzed, DNA substrate and lyses
buffer are injected in the water phase inlets. pL water in oil droplets are generated when
the water phase is broken up by the oil phase. These droplets function as micro-reactors
and ensure enhanced mixing and reaction kinetics when led though a serpentine channel
before they are collected from the outlet. Lower panel (left) shows the circularization of a
given DNA substrate that occurs in the droplets. Thereafter the droplets are dispersed and
their content exposed to a primer functionalized glass slide using a drop-trap device shown
at the upper panel (right). After RCA reaction products can be visualized in a fluorescence
microscope (middle panel, right). The lower panel shows the reaction going on the primer
functionalized glass surface, where the generated circles bind the primer. Thereafter RCA
is performed by an added polymerase giving rise to a long tandem repeat product that can
be visualized at the single molecule level.

enhancements imposed by the REEAD setup allowed for an impressive

detection limit of 0.06 parasites/mL in a single drop of unprocessed blood.
This detection limit was superior to the best PCR protocols and allowed
detection of malaria even in saliva from infected individuals [148]. Since
the development of the Plasmodium topoisomerase I specific REEAD
setup, increasing amount of REEAD assays with specificity towards
pathogen-, virus- or bacteria-specific DNA interacting enzymes have
been established [150].

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 79

3.7 Detection of Biochemical Conditions

and Small Molecules
Temperature, pH, ionic strength or various co-factors are parameters that
affect most chemical reactions and biological processes. Accurate meas-
urement of these parameters is therefore of major importance in medicine
and in most branches of science and technology. DNA has turned out to
be a prominent material for the generation of sensors that can measure
such environmental factors. One reason for this is that the DNA molecule
does not have a static structure.
In normal double-stranded DNA, the two strands are kept together by
hydrogen bonds between nucleotide bases. Depending on the base com-
position and environmental factors the DNA duplex can attain different
conformations including the common right handed B-DNA conformation
or the left handed Z-DNA conformations. Also, the bases of DNA can
stack in alternative conformations to generate triplex or quadruplex
structures. Breakage of the hydrogen bonds to separate the DNA strands
or alterations in DNA conformations can be facilitated by changes in the
surrounding aqueous solution, most notably by changes in the tempera-
ture, pH or the ionic strength of the aqueous solution. By the introduc-
tion of various modifications e.g. fluorophore–quencher pairs it has
been possible to demonstrate the functionality of a variety of DNA sen-
sors for monitoring different chemical or physical characteristics of the
surroundings.
For the generation of thermometers it has been exploited that each
duplex DNA structure in a given solution is characterized by a specific
melting temperature (Tm), which is the temperature where half of the mol-
ecules have melted and generated two DNA strands. The fraction of DNA
molecules melted at a given temperature reflects an equilibrium process
between DNA melting and re-annealing (hybridization) of the two strands.
Tm for a given DNA molecule depends on the number of hydrogen bonds
and therefore on the number and composition of base-pairs. A temperature
sensor is thus inherited in the hybridization kinetics of DNA molecules.
Consistently, nanoscale “DNA-thermometers” have been presented taking
advantage of the hybridization kinetics of DNA molecules [157–160]. So
far the DNA-thermometers have been based on the so-called “molecular

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Figure 3.4.    Schematic illustration of a DNA hairpin end-labeled with a donor–acceptor

pair of dyes. In the hairpin conformation, the acceptor (red) quenches the light emitted
from the donor (green). When the temperature increases above the melting temperature of
the hairpin (Tm) the hairpin melts, and separation of the dyes results in a fluorescent
signal.

beacons” [158] consisting of DNA molecules able to form DNA hairpin

structures. Hairpins are generated from single stranded DNA which has
complementary sequences at the ends, allowing formation of a DNA hair-
pin as a consequence of intra-strand hybridization between complemen-
tary sequences (Figure 3.4). In a DNA hairpin the duplex region generated
upon hybridization forms the stem of the hairpin and the nucleotides
between make up the hairpin loop. At a specific temperature an equilib-
rium will form between closed hairpins and single-stranded DNA mole-
cules. At temperatures below Tm of the specific DNA molecule, most
molecules will form a hairpin, but as the temperature rises above Tm the
hairpins will melt, and more molecules will be single-stranded [159].
Both the stem length and sequence of the stem is important for the Tm of
the hairpin. Moreover, the composition of the surroundings e.g. ionic
strengths and pH will affect the Tm and, hence, these factors need to be
taken into consideration when measuring temperature using DNA-based
sensors.

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 81

In the molecular beacon the hybridization kinetics is followed by

FRET between a fluorophore and a quencher located at the 5′- and 3′-end
of the DNA hairpin, respectively [158]. When T < Tm, hairpin formation
will ensure that the fluorophore is located close to the quencher in the
hairpin stem causing low fluorescence, but as the temperature increases
DNA molecules become single-stranded and the quencher is separated
from the fluorophore causing higher levels of fluorescence. The fluores-
cent signal will increase with increasing temperatures giving a sigmoid
melting curve [158–160].
The applicability of molecular beacons functionalized with terminal
fluorophore–quencher pairs as temperature sensors has been demon-
strated using a hairpin with a five base-pair stem consisting of four G–C
base-pairs with a centrally located A–T base-pair and a loop of twenty T’s
[159]. Using this sensor it was possible to follow temperature changes in
steps of 1° in the range from 37°C to 42°C and from 42°C to 32°C in
many repeated cycles by measuring fluorescence emission resulting from
melting of the beacon structure. In the same study it was demonstrated
that the temperature range in which a given molecular beacon has its opti-
mal effect as a thermometer can be changed simply by changing the base
sequence of the hairpin. Note here, that the temperature range where a
given hairpin has its optimal use as a thermometer is the temperature
range where the melting curve is steepest. The steeper the melting curve
is, the more accurate the thermometer will be, but at the same time the
range, where the thermometer can be used will be smaller. As discussed
by the authors, this can be compensated for by the simultaneous use of
structurally different hairpins, which vary in their optimal temperature
range and are coupled to different fluorophore–quencher pairs.
A separate work has demonstrated the application of molecular bea-
cons as intracellular thermometers [160]. In this study the artificial DNA-
like molecule termed L-DNA was used for the construction of the
molecular beacon that sensed the temperature. The advantage of L-DNA
for intracellular measurements of temperature is that L-DNA can neither
hybridize with natural DNA nor bind proteins, and it is resistant to intra-
cellular enzymatic degradation. These are all characteristics, which are
essential when applying the thermometer to living cells [160]. The
L-DNA thermometer was introduced into different cells that were kept at

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various temperatures, and measurements of fluorescence intensity by con-

focal microscopy demonstrated similar response curves as obtained with
buffer.
Thermal therapy where cells are killed due to a high local temperature
increase is a promising method for cancer treatment [161]. However, this
method has been hampered by the lack of reliable means to measure the
temperature in the organism at the single cell level. The L-DNA thermom-
eters have been used to monitor temperature inside cells that have been
added Pd nanosheets [162] and irradiated with a 808 nM laser to increase
the intracellular temperature. In these experiments the L-DNA thermom-
eters were able to give reliable measurements of the intracellular tempera-
ture. These thermometers may therefore be valuable tools in future
treatment of cancer using thermal therapy.
As mentioned above, besides temperature, hybridization of nucleotide
bases depends on the ion strength and pH of the surrounding aqueous
solution [159]. A higher ionic strength and a higher pH increase the stabil-
ity of the DNA double-helix due to a partly neutralization of the repulsive
electrostatic interactions between the phosphates in the DNA strands. In
the work with the L-DNA thermometers, it was demonstrated that the
melting curves of the used hairpins did not change very much under the
normal intracellular variations in ionic strength and pH, which have been
suggested to vary between 150 and 200 nM of monovalent salts and pH
6.8 to 7.4 [160, 163, 164]. However, in the work by Johnstrup et al. a
severe effect was observed when the ionic strength was increased from
0  to 1000 mM of NaCl. As suggested by the authors the ionic strength can
be used to fine tune DNA thermometers to obtain a desired temperature
range or alternatively, DNA hairpins can be used to directly measure the
ionic strength in solutions, where the temperature is kept constant. In the
same way DNA hairpins can be used as pH-meter in situations, where
temperature is kept constant [159].
Other examples of sensors for monitoring pH in living cells have been
presented by the Krishnan group who described the utilization of a mis-
matched duplex with a functional C-rich domain that forms an intramo-
lecular i-motif at acidic pH. In this setup donor and acceptor fluorophores
inserted in the C-rich domain were used as reporters for pH dependent
conformational changes [165]. By changing the sequence of the pH

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sensitive domain of this type of sensors it was possible to tune the pH

sensitive regimens and a family of sensors spanning ranges from pH 4
to 7.5 was created [166]. The sensors were used to probe pH of early
endosomes and the trans-Golgi network in the same cell and in real time
[167]. Moreover, by fusion to recombinant antibodies it was possible to
deliver the pH sensors along the endocytic pathways to specific organelles
[168, 169]. In another design Amodio et al. [170] utilized triplex-based
DNA strand displacement strategies that can be triggered and finely regu-
lated at either basic or acidic pHs to monitor pH in the surrounding solu-
tion, while aggregation of poly-dA functionalized gold-nanoparticles by
pH dependent A-motif formation allowed a colorimetric readout of
pH [171].
On top of the above mentioned pH sensors numerous nanoswitches
or — sensors have been designed to measure the chemical composition
of their surroundings. These include a mechano-electronic DNA switch
designed by the Sen group [172]. This switch binds Hg2+ in T–T mis-
match regions in a manner that leads to a conformation change, which
can be measured by FRET. In one of the earlier examples Seeman and
coworkers used the transition between the B and Z form of DNA in a
rigid double-crossover molecule to monitor the chemical composition in
the surrounding buffer [173].
The identification of increasing numbers of small molecule biomark-
ers with relevance for treatment or diagnosis of human diseases [174–177]
have led to the development of an increasing number of assays for detec-
tion of such analytes. Oligonucleotides with unique target-recognition
elements, namely the so-called aptamers, have been introduced [92–
94, 178]. Among other things, aptamers have been used as key-lock sys-
tems for nanocarriers and they were demonstrated to facilitate release of
a reporter molecule from a DNA icosahedron upon binding of a small
molecule chemical trigger (cdGMP) [179].
Aptamers can also be combined with the above mentioned RCA based
signal enhancement methods. Indeed, a promising family of assays for
detection of biomolecules in solution takes advantage of clever designed
structure-switching aptamers that can be circularized and thereby tem-
plate RCA only upon target binding. One example on this was the RCA
based detection of the small molecule ATP by Cho and coworkers [96].

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84 B. R. Knudsen et al.

Table 3.1.  DNA sensors for the detection of various kinds of biomolecules and
­biochemical conditions.
Signal
Transduction
Assays DNA Probes Mechanisms Timescale References

DNA/RNA
DNA ssDNA Optical Equilibrium [50–62]
RNA (cDNA) ssDNA Optical and Equilibrium [55, 63–67,
electrochemical 73–76,
88–91]
Proteins
Proteins ssDNA Optical and Equilibrium [98, 99, 
(aptamer) electrochemical 101, 
and 104–111]
ssDNA
conjugated
antibodies
Enzyme Activities
Endonuclease ssDNA Optical Real time [115–120]
detection dsDNA
DNA polymerases dsDNA Optical Real time [122]
DNA repair ssDNA Optical Real time [123–126]
activities dsDNA
DNA ssDNA Optical and Real time [127–130]
topoisomerases dsDNA electronic
DNA ssDNA Optical and Equilibrium [145, 146, 
topoisomerases mechanical 148]
and recombinases
Biochemical Conditions
Temperature ssDNA Optical Real time [159, 160]
pH ssDNA Optical Real time [165–167, 
ssDNA 170]
Ions dsDNA Optical and Real time [172, 173]
electronic
Small molecules ssDNA Optical [96, 179]

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DNA Sensors for the Detection of Biomolecules and Biochemical Conditions 85

3.8  Conclusion and Future Perspective

The development of DNA nanosensors is currently a vibrant area which
has shown significant promise on detecting an array of biomolecules and
biochemical conditions, as outlined in Table 3.1. Aside from the excite-
ment of utilizing DNA sensors in studying fundamental reaction kinetics
or single cell characteristics, there exist a multitude of opportunities for
promoting DNA nanosensors into clinical diagnostics. However, it is
imperative to consider the challenges of maintaining the functionality in
biological crude samples, and the ease of detection. Most DNA nanosen-
sors have been validated in the absence of body fluidics where there may
be non-specific biological components interfering to the sensor specificity
and sensitivity; The emerging detection approaches through mechanisms
of, for example SPR, scattering, or surface enhanced Raman scattering,
are highly sensitive and specific, however, highly dedicated equipment is
often required and is thus not suitable for the clinical setting. As the field
evolves, there will be undoubtedly a great number of strategies developed
to conquer the above-mentioned challenges, and we may see DNA
nanosensors entering the clinics in the foreseeable near future.

Acknowledgements
The authors would like to acknowledge the support from the Start Up Fund
and the Direct Grant provided by the Chinese University of Hong Kong.
We would also like to thank Dr. Sissel Juul Jensen in granting the use of her
results in Figure 3.3.

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Chapter 4
.

DNA Nanostructures in Cell

Biology and Medicine

Liqian Wang*,‡ and Giuseppe Arrabito†

*Laboratory of Physical Biology

Shanghai Institute of Applied Physics
Chinese Academy of Science, Shanghai 201800, China
†
Department of Physics and Chemistry
University of Palermo, Viale delle Scienze, Parco d’Orleans II
90128 Palermo, Italy
‡
wangliqian@sinap.ac.cn

This chapter gives a clear description of DNA nanostructures in physio-

b

logical environments and the mechanism of them entering cells. This

chapter also summarized the most advanced DNA nanostructures for drug
delivery and other applications, such as cell- systematic evolution of
ligands by exponential enrichment (SELEX), dip-pen nanolithography
(DPN) and, etc.

99

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100 L. Wang and G. Arrabito

4.1 Introduction
DNA nanotechnology has enabled a new idea of building smart therapeu-
tic nanodevices and drug delivery systems which has several advantages
over conventional ones. In conventional nanomedicine, people tend to use
inorganic materials, which are toxic and difficult to degrade in human
body [1–3]. On the other hand, artificial DNA nanostructures are biocom-
patible, highly stable and programmable. As the fact that DNA is existed
.

in human cells, the cells have undergone millions of years of evolution to

learn to use, to process and to synthesis DNA. This special bond between
DNA and cells makes DNA an ideal material in cell biology and medicine.
DNA nanostructures also has other unique properties, such as precise
structural assemblage at 1D, 2D and 3D. Previous studies have suggested
the optimal size of a drug carrier is between 20 and 100 nm [4–6]. When
nanoparticle reaches more than 20 nm, it can avoid renal clearance and
enhance drug delivery, thus the high loading capacity of DNA structural
based nanocarriers can escape renal clearance and also they are small
enough to get into tumor regions [7]. DNA nanostructures are noticed to
be an ideal drug delivery carrier not only because of the size, but also
because DNA nanostructures can carry multiple drugs at one time and
control multiple drug releases. In this chapter, we summarize the recent
progress towards the idea of bringing DNA nanostructures into cells; we
discuss the stability of DNA nanostructures in cellular environment and
most advanced DNA nanocarriers for drug delivery. We begin with how
DNA aptamer recognize live cells and then manipulate single cell by
DNA–protein immobilization.
b

4.2  Cell-SELEX for Cellular Detection

The ability to detect and understand changes in cell conditions at a molec-
ular level is of fundamental importance for the accurate diagnosis and
timely therapy of diseases. As previously discussed in Chapter 2, DNA
aptamers are excellent candidates as molecular probes able to recognize
extracellular matrix signatures of cancer cells and target cell-specific
ligands for therapeutic purposes. The selection process for DNA aptamers
able to recognize live cells is defined cell-SELEX [8].

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DNA Nanostructures in Cell Biology and Medicine 101

The process begins with the preparation of a oligonucleotide library

and the growth of target cell cultures. Target cells are incubated with the
DNA pool, cell-bound oligonucleotides are collected in order to generate
a new enriched pool through the amplification of bound oligonucleotides.
Incubation of DNA pool on control cells (counter-selection) is performed
in order to reduce non-specific binding. Aptamer binding affinity towards
target cells is quantified by flow cytometry. The selection process of oli-
gonucleotide sequences is repeated until the binding assay indicates
.

enough affinity and specificity to the target cells (typically 10–20 rounds
are performed). The last selected DNA pool is cloned in Escherichia
coli, and sequenced in order to generate candidate sequences. These are
selected, synthesized and applied to binding assays. DNA aptamers have
to be optimized my minimizing length (in order to reduce cost) and maxi-
mizing binding affinity. Prediction of secondary structures is carried out
by the program mfold (http://unafold.rna.albany.edu/?q=DINAMelt). By
comparing binding affinities among selected DNA aptamers belonging to
the same pool, critical sequences for target binding are predicted [9].
DNA aptamer selection on cells was firstly performed against whole
cells [10]. At that time — i.e. 2003 — aptamers involved cancer detection
were limited by the absence of aptamers targeting cancer cell membrane
proteins. By 2003, few cancer biomarkers had been identified. Prof. Tan
group established a new strategy of selecting aptamers specific to whole
live cells. Notably, phenotypic variations between cell types, such as
between normal and cancer cells give rise to differences in molecular
signature. Therefore, the ability to isolate aptamers based on these molec-
ular signatures permits to create molecular probes specific to cancer cell
types for use in cancer diagnosis and therapy. This process, known as cell-
b

based SELEX, or cell-SELEX, was first developed using a human acute

lymphoblastic leukemia cell line, CCRF–CEM (T-cell line), as the target
cell [11].
The result of the process was the generation of a panel of aptamers
specifically binding target cells, including one able to bind to a membrane
protein tyrosine kinase 7 — a biomarker for leukemia [12].
DNA aptamers have been used to build targeting DNA nanostructures,
aptamers–drug conjugates and smart DNA diagnostic/therapeutic net-
works (see Figure 4.1) [13]. Examples of applications in intracellular

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102 L. Wang and G. Arrabito

ssDNA library
Target cells

Sequencing
Cloning
Positive selection wash Remove
.

Evolved unbound DNA

DNA pool
cell SELEX
PCR
amplification

Extract
bound DNA
Retain
Unbound DNA
Counter
selection Negative
Remove Cells
Unbound DNA

Figure 4.1.    Schematic representation of cell SELEX process. Reproduced in part with
­permission of The Royal Society of Chemistry from Ref. [13].

biomolecular imaging by engineered DNA nanostructures containing

aptamers have been shown for ATP by Fan et al. [14]. or mRNA by Tan
et al. [15].
One of the most interesting applications of DNA aptamers is the
detection of cancer cells, in particular circulating tumor cells, which leak
from a metastasis into blood vessels, and transfer to distant locations, with
b

the potential to establish metastatic tumors [16]. Remarkably, the sensitive

detection of cancer cells plays a critical role in the early diagnosis of can-
cer and eventually allows for separation form benign cells. As examples
of DNA aptamers integration in cell sensing devices, Qu et al. [17].
designed an electrochemical sensor by using two cell-specific aptamers,
TLS1c and TLS11a which recognize MEAR cancer cells, with the possi-
bility to detect as few as a single MEAR cell in 109 blood cells. Li et al.
[18]. showed an aptamer functionalized hydrogel that could catch cancer
cells with a density over 1000 cells/mm2.

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DNA Nanostructures in Cell Biology and Medicine 103

Interestingly, DNA aptamers have also been shown to constitute fun-

damental tools for novel DNA-based therapies. We here briefly discuss
about integration of DNA aptamers in nanostructures based on DNA
usable in chemotherapy, gene therapy and immunotherapy.
Given their high loading efficiency and cell type-specific recogni-
tion capability, DNA nanostructures can improve distribution and effi-
cacy of chemotherapeutic drugs in vitro and in vivo [19]. Many
chemotherapeutic agents have been conjugated with DNA to form cova-
.

lent or non-covalent conjugation for chemotherapy (see e.g. Ref. [20]).

Aptamer-integrated DNA nanostructures have been developed for tar-
geted delivery of therapeutic drugs to reduce side effects as shown by
Zhu et al. [21] in his aptamer-tethered DNA nanotrains (aptNTrs) for
targeted chemotherapy.
Gene therapy is considered one of the most promising approach for
treating diseases such as cancer or viral infections. In this therapeutic
approach, small interfering RNA (siRNA) and microRNA (miRNA) mol-
ecules are employed as gene silencing tools. The bottleneck of this
approach is constituted by the efficiency of gene delivery vectors. In this
sense, DNA nanostructures have been explored as suitable non-viral vec-
tor candidates because of their high payload capacity and excellent bio-
compatibility, flexibility and mechanical stability. Examples of integration
of DNA aptamers within DNA-based nanostructures usable as vectors are
shown by Giangrande et al. [22]. who used aptamer–siRNA chimeric
RNAs to deliver functional siRNAs to target cells or by Zhang et al. [23].
who constructed an aptamer–siRNA complex able to deliver siRNA
in vivo, in which aptamers and siRNA are hybridized to the outermost
layer of DNA dendritic nanostructures.
b

DNA nanostructures have been reported to conjugate with vaccine-

based, cytosine-based guanine oligodeoxynucleotide (CpG)-based immu-
notherapies to target cancer cells. CpG motif have demonstrated to be an
immunotherapeutic adjuvant in the treatment of a wide range of diseases,
since it can stimulate dendritic cells, B cells, and macrophages through
interaction with the toll-like receptor 9 (TLR9) [24]. For example, Liu
et al. [25] employed a tetrahedral DNA nanostructure as a scaffold to
assemble a model antigen and CpG adjuvants together into nanoscale
complexes. In cell-based immunotherapy, killer lymphocytes have been

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104 L. Wang and G. Arrabito

used to kill cancer cells. However, these cells have to be targeted to cancer
cells. DNA aptamers anchored on the cell surface of leukemia cell lines
have been shown by Xiong et al. [26] to improve immune-efficacy of
these cells and cytotoxicity towards cancer cells. Although the potentiality
of DNA aptamers in cell biology has been shown, there are still some
limitations that hamper applications in biomedical field such as the
lengthy process of cell-SELEX and the technical difficulty in the precise
identification of aptamer targets, since they are often constituted by mem-
.

brane proteins.

4.3  Single Cell Manipulation by DNA-directed

Protein Immobilization
One of the most intriguing challenges in modern bioanalytical sciences is
the rapid and accurate identification of a large number of different molec-
ular species with high temporal and spatial resolution [27]. This analytical
capability is of outmost importance in the characterization of cellular
samples at the single cell level — allowing for truly molecular quantitative
analysis. In fact, any cell phenotype is determined by biomolecular net-
work states maintained by the dynamics of multiple molecular interac-
tions. The effect of a drug molecule is, basically, based on the perturbation
of molecular interactions in such system. The capability to quantitatively
analyze a single cell is then crucial for understanding the precise cellular
state, especially in the case of medically relevant conditions such as
necrosis, apoptosis, proliferation, differentiation, transformation, senes-
cence, growth or shrinkage which are all associated with a profound per-
turbation of the cytoplasmic content.
b

4.3.1  DNA-directed immobilization

The employment of surface-based bioanalytical tools like microarrays has
allowed for breakthrough in fundamental and applied biomedical research,
in the form of DNA and protein microarrays. For better understanding
complex cellular features in healthy or diseased states, researchers can
use  microarrays to delineate on a molecular level the characteristics of
individual cells within complex mixtures of cells belonging to different

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DNA Nanostructures in Cell Biology and Medicine 105

populations. As a consequence, it is possible to quantitatively identify

distinct cellular types/states according to the expression of particular cell
surface molecules, and also by analyzing their response to precise signals
through the secretion of factors or cellular activities. In this regard, it
becomes of fundamental importance to establish an efficient immobiliza-
tion strategy, which allows for oriented immobilization of small molecules
and protein molecules usable for capturing cells. The main issue in this
regard is constituted by the ability to retain the biological functionality of
.

the immobilized biomolecule in order to be able to function as capture

probes.
In this regard, DNA-directed immobilization (DDI) of oligonucleotide-
tagged components on DNA microarrays bearing complementary oligo-
nucleotides is a powerful and chemically mild process, which fulfils the
aforementioned requirements. The DNA-directed immobilization method
[28] takes advantage of specific hybridization of complementary oligonu-
cleotides and thereby allows the site-specific capture of biomolecules by
the DNA microstructures on a solid substrate (see Figure 4.2).
b

Figure 4.2.  Scheme of DDI. Reprinted with permission from Ref. [28]. Copyright
(2014) Elsevier.

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106 L. Wang and G. Arrabito

In addition to numerous applications in the area of protein chips for

diagnostics [29], the DDI method has also been employed for the genera-
tion of bioarrays of living cells [30].
Reisewitz et al. [31] firstly showed the possibility to build up specific
cellular capture platforms by DNA directed immobilization of specific
cellular antibodies, i.e. the cell specific mouse-anti human CD81 IgG
(αCD81). They employed a biotinylated IgG coupling it with covalent
conjugates of STV and ssDNA oligonucleotides which were hybridized
.

with a DNA microarray containing complementary capture oligonucleo-

tides, spotted by piezoelectrical inkjet printing on modified glass surfaces.
The resulting antibody array was employed for the adhesion of HEK293
cells and Hodgkin lymphoma L540cy cells. Remarkably, the adhered
cells showed viability and ability to propagate to form a densely grown
monolayer, restricted to the lateral dimensions of the DNA spots inside the
array. The authors showed the feasibility of their approach by employing
two different set-ups, static incubation and hydrodynamic flow inside a
microfluidic channel. This last approach permitted to execute cellular
capture and selection of cells from culture medium. The microfluidic
polymer chip consisted of PDMS substrate containing a microfluidic
channel and inlet/outlet ports, mounted on a top a glass slide bearing a
DNA microarray to enable IgG binding by DDI. The authors observed
that it was possible to obtain optimal cellular capture at flow rates as high
as 1 µL/min, whereas a higher flow rates determined significant decrease
of immobilization efficiency.
Given the possibility to capture cells to DNA modified surfaces, the
possibility to employ DNA arrays as capture matrix can permit the devel-
opment of the single-cell biochips in which a single cell is immobilized
b

on an array containing different microspots with lateral dimensions which

are smaller than the lateral size of a single cell. This would permit quan-
titative analysis at the single cell level.

4.3.2  Dip-pen nanolithography coupled with DDI

for single cells interaction arrays
Single cell microarrays require features smaller than 10 μm for functional
manipulation of sub-cellular structures. Several top-down methodologies

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DNA Nanostructures in Cell Biology and Medicine 107

.

Figure 4.3.  (a) Fabrication of microarrays by Dip Pen Nanolithography for DNA-

directed protein immobilization and subsequent generation of live-cell arrays for the
recruitment of transmembrane Epidermial growth factors (EGFR) receptors on MCF 7
cells. Reproduced from Ref. [32]. Copyright © 2013 by John Wiley and Sons, Inc.
Reprinted with permission from John Wiley and Sons, Inc. (b) Scheme of generation of
multiplexed antibodies microarrays by DDI. (c) Bait- PARCs displaying VSV-G epitope
tags are recruited to anti-VSVG functionalized surface patterns within the plasma mem-
brane of COS7 cells. Scale bar is equal to 5 microns. Reproduced from Ref. [33].
Copyright © 2014 by John Wiley and Sons, Inc. Reprinted with permission from John
Wiley and Sons, Inc.
b

like electron beam lithography and microcontact printing can be employed

for indirect surface patterning at this scale, however, those approaches
often require clean rooms and multiplexing of several different biomole-
cules on the same surface is limited [27]. In this regard, Arrabito et al.
combined DPN and DDI of proteins to fabricate cell-compatible function-
alized glass surfaces (see Figure 4.3) [32].
DPN was used to realize multiplexed patterns of capture-oligonucleotides
onto activated glass surfaces. In general, DPN allows for direct deposition

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108 L. Wang and G. Arrabito

of molecules from an ink-loaded tip onto a surface. One has to consider

that the diffusive or liquid aggregation state of the ink determines deposi-
tion mechanism. In fact, in the diffusive case, molecules are dispensed to
the surface through a water meniscus that forms naturally when the tip is
brought in contact to the surface. Such process is dependent on molecu-
lar diffusion rate and it is reported to be slow for DNA molecules (10–12–
10–13 m2/s). On the other hand, in the case of liquid inks, ink molecules
remain homogeneously dissolved in the carrier solution. When the ink-
.

loaded pen contacts the surface, a liquid meniscus is formed, which is

composed of the liquid ink itself and water from the atmosphere. By pull-
ing the tip away from the surface, the meniscus breaks and the spot is
formed. In the liquid ink mechanism, the deposition process depends on
the relative humidity (i.e. the amount of water vapor present in air
expressed as a percentage of the amount needed for saturation at the same
temperature) surface tension in between liquid and tip, liquid and surface
as well as on the liquid carriers’ viscosity. Remarkably, the deposition
process is not dependent upon molecular weight of the deposited biomol-
ecule, so it is suitable for depositing proteins and DNA molecules. On the
other hand, it is dependent on the physical–chemical properties of the
liquid carried in which the DNA is dissolved.
In this regard, Arrabito et al. optimized a strategy that employs oligo-
nucleotide Dip-pen Nanopatterning of oligonucleotide dissolved in
Polyethylene glycol (PEG) matrixes. PEG is a water-soluble and biocom-
patile polymer commonly used as an additive in solutions for biological
applications. Complementary sequences conjugated to a protein of inter-
est were hybridized to the spotted sequence via DDI. A systematic study
on the parameters affecting printing efficiency was performed. Parameters
b

like oligonucleotide concentration, molecular weight of PEG, dwell time,

relative humidity were investigated in order to derivate the best conditions
for hybridization efficiency and array writing throughput. The results of
these studies was that the best conditions between small feature sizes and
high density of immobilized oligonucleotide are obtained by PEG 1000
inks showing relative humidity as high as 30%, with a capture oligonu-
cleotide concentration of 100 µM.
Oligonucleotides complementary to the immobilized capture
­oligonucleotides were covalently linked to streptavidin, and the resulting

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DNA Nanostructures in Cell Biology and Medicine 109

conjugates were functionalized with biotinylated antibodies and fluoro-

phores. These streptavidin–antibody complexes then bind to the immobi-
lized capture-oligonucleotide arrays. In particular, the produced arrays
were functionalized with epidermal growth factor (EGF) taking advantage
of covalent ssDNA–streptavidin conjugates as adaptor molecules. The
surface-immobilized EGF was used for recruiting EGFR in the plasma
membrane of MCF7 cells.
The combination of DPN with DNA directed immobilization has
.

also been shown for the fabrication of multiplexed patterns of capture-

oligonucleotides onto activated glass surfaces — this meaning that it was
possible to generate microscale arrays of two capture oligonucleotides,
corresponding after DDI to two different proteins, at dimensions below
single cells [33]. This was possible by inking Dip-pen arrays with two
different oligonucleotides by a microfluidic inking device. At the outset
the first sub-array is written, then after a translation along the X-axis and
a pens microscale alignment on the previous pattern, the double-oligonu-
cleotide array is completed with the second sub-array. The pen array is
translated along the X-axis of 66 µm to the right and along the Y-axis of
100 µm on to write new arrays.
This multiplexed microarray methodology was applied to simulta-
neously measure the interaction of two bait-presenting artificial recep-
tor constructs (PARCs) — i.e. the regulatory domain RII-b and the
regulatory domain RI-a of protein kinase A (PKA) — with the prey
protein construct constituted by the catalytic subunit mCherry-cat-α of
PKA fused to the fluorescent protein mCherry inside individual living
cells. The two different PARCs were orthogonally attached to the sur-
face since in their extracellular region, they showed two different
b

epitopes, respectively VSV-G bait and HA bait that are selectively

captured by respective biotinylated antibodies linked to the ssDNA–
streptavidin conjugate which is hybridized to the complementary
ssDNA deposited by DPN. In Fig. 4.3, we show a single cell recruited
on an anti-VSVG functionalized surface. For this, micrometer-scale
arrays of antibodies with binding specificity for the peptide epitope on
the bait-PARC were generated by combining DPN and DDI. It was
possible to successfully discriminate the interaction of RII-b and RI-a
with mCherry-cat-α [33].

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110 L. Wang and G. Arrabito

4.3.3  High-throughput patterning methodologies

for single cells interaction arrays
To improve the efficiency of multiplexed surface patterning, Arrabito et al.
developed a prototype of a robust custom plotter based on 2D polymer-pen
lithography (2D-PPL), a patterning methodology in which a soft elasto-
meric polymer (polydimethylsiloxane) delivers inks onto a surface by
controlling the movement of the pen array with a scanning probe micro-
.

scope [34]. A typical polymer pen array contains thousands of pyramid-

shaped tips made with a master prepared by conventional photolithography
and subsequent wet chemical etching. These pyramids are connected by a
thin PDMS backing layer (thickness around 50−100 nm) that is adhered
to a glass support [35]. This device enables rapid fabrication of microar-
rays at ambient conditions in a multiplexed direct-writing mode. The
printing process was carried out by polymeric pyramidal pens onto which
multiple (up to 36) ssDNA solutions can be loaded through a microfluidic
inkwell device. Subsequent to optimization of ink viscosity and surface
tension by glycerol and tween-20, DNA arrays were plotted and used for
DDI of EGF-bearing ssDNA–streptavidin conjugates. The resulting
microarrays covered areas of about 0.5 cm2 using up to three different
oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of
15 μm, leading to densities of up to 104–105 spots/mm2.
The microarrays were capable of recruiting and activating EGF recep-
tors in sub-cellular regions within human MCF7 cells. In order to verify
if immobilized EGF was able to activate EGF receptors, cells were stained
with antibodies specifically directed against active EGFR, which is phos-
phorylated at tyrosine 1068. The ratio between phosphorylated and total
b

EGFR was used to measure the activation state of this receptor. In this
sense, the authors found out that a significantly higher fraction of EGF
receptors was phosphorylated within cell regions that contact EGF func-
tionalized surfaces. Such results confirm the ability of Polymer Pen
Lithography to produce functional sub-cellular scale EGF arrays able to
activate EGF receptors in cells (see Figure 4.4).
In a very recent breakthrough paper, Angelin et al. [36]. demonstrated
the site-directed sorting of differently encoded, protein-decorated DNA-
origami structures on DNA microarrays. The combination of bottom-up

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DNA Nanostructures in Cell Biology and Medicine 111

immobilized total EGFR phospho-Y1068 ratio phospho/total

EGF (TIRF) EGFR (TIRF) EGFR
EGF immobilized via soft-pen

4
arrays and DDI

0
.

10µm

Figure 4.4.    Experimental evidence of EGF Receptor activation by surface immobilized

EGF ligands. EGF was immobilized on oligonucleotide arrays printed on glass substrates.
To visualize successful immobilization, the DNA–STV–biotin–EGF complexes contained
biotin-Atto740. MCF7 cells expressing EGFP–EGFR were cultured on those functional-
ized surfaces and visualized by Total internal Reflectance Fluorescence microscopy.
Immunostaining using phosphor-specific revealed increased phosphorylation of EGFR at
Tyrosine residue 1068 in subcellular regions contacting spots with immobilized EGF.
Reproduced from Ref. [34]. Copyright © 2014 by John Wiley and Sons, Inc. Reprinted
with permission from John Wiley and Sons, Inc.

self-assembly of protein–DNA nanostructures and top-down micropat-

terning — carried out by polymer pen lithography–of solid surfaces ena-
bled the creation of multiscale origami structures as interface for cells
(MOSAIC). They designed a rectangular origami construct from the 5438
nucleotides template 109Z5 having nine single-stranded DNA (ssDNA)
binding tags, protruding from one side of the plane of the quasi-2D nano-
structure. The nine binding sites are able to bind to their complementary
surface-bound capture oligonucleotides.
b

This innovative technology was applied to investigate the activation of

EGF receptors in living MCF7 cells through distinctive nanoscale arrange-
ments of EGF ligands. This technology can represent a significant break-
through in bioanalytical applications since it permits the assembly of
structures having the same size of biomolecular assemblies present in the
membrane of living cells. Such assemblies are composed of tens to thou-
sands of molecules (sizes around 5–100 nm) and play a crucial role in
the outcome of signaling events and the development of the cellular
phenotype.

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112 L. Wang and G. Arrabito

4.4  How to Get into Cells

To get into cells, we firstly need to understand the entries. Such as
human civilization that if we want to export our goods to foreign coun-
tries we need to know the location of the ports, cultures and policies.
Similar situation happens to cells, they must be accessed through entry
points, and this is related to “protector” of cells — membranes. The
membrane encloses the cell and maintains the essential differences
.

between intercellular and extracellular environments. Inside cells, there

are organelle membranes that do the same job to keep each organelle in
different environment than cytosol. So, membranes are critical and una-
voidable entrees for big or small molecules, as well as DNA nanostruc-
tures. In this part, we will discuss the different regulated modes of
getting into cells.
The entry processes for macromolecules (proteins and DNA nano-
structures) and small molecules (ions and amino acids) to get into cells
are different. For macromolecules, they are transported into cells under
a number of active and regulated processes that is called endocytosis
(endo “within” cytosis “cell”) [7, 37]. It is a process in which a macro-
molecule gets into a cell without passing through the cell membrane.
Usually, the endocytosis process falls into two categories: phagocytosis
and pinocytosis (Figure 4.5). These two categories can be mainly distin-
guished by the size of the object ingested. Mammalian phagocytosis is
limited to particular cells, such as macrophages, monocytes, dendritic
cells and neutrophils; however, pinocytosis happens to all cells through
four endocytic uptake pathways: macro-pinocytosis, clathrin-mediated
endocytosis (CME), caveolae-mediated endocytosis, and clathrin/caveo-
b

lin-independent endocytosis. The relationships among those processes

are shown in Figure 4.5.

4.4.1  Phagocytosis
Phagocytosis, which means “cell-eating,” is the process in which cells
swallow large objects. As shown in Figure 4.5, during phagocytosis, the
membrane first folds around the target, and then the object are sealed into

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DNA Nanostructures in Cell Biology and Medicine 113

Phagocytosis Pinocytosis

Macro-pinocytosis
Clathrin-mediated
endocytosis
Caveolae-mediated
endocytosis
Clathrin/caveolin-independent
endocytosis
Specific receptors
.

>1 µm >1 µm ~120 nm ~60 nm ~90 nm

Figure 4.5.    Multiple gateways into living cells. Different endocytic mechanisms use dif-
ferent sized endocytic vesicles to bring in a variety of cargoes.

large vesicles. The large vesicles usually called as phagosome, which is

generally larger than 250 nm in diameter. Usually, the defense organisms
against non-self intruders, such as bacteria, viruses and drug delivery
nanostructures, trigger phagocytosis [38]. The process starts with the
recognition of target particles and the non-adhesion of opsonized parti-
cles to the macrophage, and then the macrophages ingest the particles
[39]. During the process, opsonized particles are connected to the mac-
rophage through specific receptor-ligand interactions, which will trigger
b

actin assembly and swallow-up of the target particles. Ultimately, the

phagosome fuses with the lysosome to form phagolysosome. This is
where the degradation of endocytosed particle goes. There is one directly
properties of ingested particles that affect the rate of phagocytosis: size.
Usually, when particles are smaller than 250 nm in diameter, they are less
effectively internalized [40]. The size of the particles in serum always
affect the extent of opsonization; therefore, influence the rate of
­phagocytosis [41].

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114 L. Wang and G. Arrabito

4.4.2  Pinocytosis
Pinocytosis is another category of endocytosis, which literally means
“cell-drinking.” Different from phagocytosis, this process involves the
uptake of fluids and using small vesicles. As mentioned before, this pro-
cess can be split to four uptake pathways:

4.4.3  Macro-pinocytosis
.

Macro-pinocytosis is similar to phagocytosis, it takes big particles and it is

driven by actin. The difference is the actin-driven membrane protrusions do
not fold around the targets but collapse onto and fuse with the plasma mem-
brane and then forming large macropinosomes with sizes comparatively to
phagosomes (Figure 4.6(a)). The intracellular destiny of macropinosomes
varies with cell type, but most commonly these macropinosomes will fuse
with lysosomes and be observed as acidification and shrinkage [42].

4.4.4  Clathrin-mediated endocytosis

CME is claimed as the major route for endocytosis in most cells. CME can
be divided into two types: receptor-dependent and receptor-independent.
Both types are also internalizing target particles and digesting for degra-
dative lysosomes. Receptor-dependent CME is a very common pathway
for ligand–receptor complexes to get through [43], thus it is also a very
important pathway for drug delivery carriers that aim at these receptors.
CME happens in the membrane region that is clathrin-enriched. Clathrin
is mainly cytosolic coat protein, during CME it deforms the membrane
b

into a coated pit and then leading to a ~120 nm acidified early endosome
which ultimately will fuse with a prelysosomal vesicle containing
enzymes leading to a late endosome. The late endosome will turns into a
lysosome at the end [44] (Figure 4.6(b)). If the target is aiming at the
cytosol, our drug carriers must escape from the endosome fast enough to
avoid lysosomal degradation. The other type of CME, the receptor-inde-
pendent CME, has a slower internalization rate compares to the other type
[45]. During this type of process, the particles usually interact with the
plasma membrane via non-specific charges or hydrophobic interactions.
However, the destiny goes the same way as receptor-dependent CME.

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DNA Nanostructures in Cell Biology and Medicine 115

(a) Macropinocytosis (b) Clathrin-mediated (c) Caveolae-mediated

endocytosis endocytosis
.

Clathrin- Caveolar
coated vesicle
Macropinosome vesicle

Early
endosome Caveosome

Late
endosome
H+
Endoplasmic
reticulum
Golgi
Lysosome
Nucleus

Figure 4.6.  Diverse intracellular fates of carriers after different endocytic uptakes.

(a) Macropinocytosis forms macropinosomes of large cargos, which are subsequently
fused with lysosomes. (b) CME gives rise to an early endosome, which is acidic, followed
by a late endosome after fusing with pre-lysosomal vesicles containing enzymes (red vesi-
cle), and finally coalesces into acidic lysosomes, which contain high levels of degradative
enzymes. (c) Caveolae-mediated endocytosis leads to the formation of a caveosome, pos-
sibly bypassing enzymatic degradation of the endo-lysosomal pathway. Reproduced with
permission from Springer from Ref. [42].
b

4.4.5  Caveolae-mediated endocytosis

Caveolae are small (~60 nm) flask-shape pits in the membrane that resem-
ble the shape of caves (Figure 4.6(c)). They are made of cholesterol-
binding protein caveolin with a bilayer full of cholesterol and glycolipids.
Besides the clathrin, they are the second most common reported plasma
membrane buds. They exist on the surface of many cell types, especially
in endothelial cells and adipocytes. CvME is an extremely regulated

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116 L. Wang and G. Arrabito

process that involves multifaceted signaling pathways. The interesting

point of CvME is that the particles being internalized could be the main
driver of the whole process [44]. The caveolar vesicles from the separation
of caveolae from the plasma membrane do not contain any harmful
enzymes, so it is noticed that many bacteria and viruses adores this path-
way just to avoid enzyme degradation. Thus, it is worthwhile to exploit
this mechanism for the research of DNA nanostructures evasion of degra-
dative CME pathway [46]. Furthermore, the caveolar vesicle does not
.

have to go through the endo-lysosomal pathway, it can go to the cave-

some. We can say CvME may open a window of opportunity for particles
to escape from endosome, which leads to create drug carriers that escape
the lysosomal degradation pathway [42]. But in reality, most cells has
slower internalization rate through CvME than CME. And the carriage
capacity of cavelolar vesicle can only load low fluid-phase volume [44].

4.4.6  Clathrin- and caveolin-independent endocytosis

There are also other small structures like caveolae on the surface of
plasma membrane, which is call “lipid rafts.” Usually these small struc-
tures have a diameter of 40–50 nm and feely lay on the cell surface [47].
These “lipid rafts” can be internalized within any endocytic vesicles. This
process happens in all type of cells. Unfortunately, the exact mechanisms
governing these types of endocytosis are still mysteries; thus we would
like to encourage people to exploit this beautiful unknown area.

4.5  DNA Nanostructures in Cellular Environment

b

The potential of DNA nanostructures to work as drug delivery cargo is

related to its structural stability in cellular environment. When we perform
the assemblage of DNA nanostructures, we usually do it under room tem-
perature in sterile conditions with appropriate buffer and salt concentra-
tions. However, when the DNA nanostructures goes into blood or even
into cells, the environmental conditions are more complicated. In vivo,
DNA nanostructures will face DNases, higher temperature than room
temperature, low salt concentrations (especially Mg2+ concentrations
are usually lower than 1 mM) and multiple different opsonins and DNA

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DNA Nanostructures in Cell Biology and Medicine 117

binding proteins. All these complex conditions make it hard for DNA
nanostructures to work properly as drug delivery cargo.

4.5.1  DNA structures in cell lysates

Cell lysates are mixtures of cellular components that have been homoge-
nized. Lysates does not contain any cell wall, so DNA structures can eas-
ily get into a similar environment as inside the cell. According to the
.

research followed by Yan and colleagues that DNA origami can stay in
cell lysate and could be extracted and characterized after 12 h of incuba-
tion [48]. However, long single/double-stranded nucleic acids cannot be
recovered after incubation. Thus, DNA nanostructures are more stable
than DNA strands in lysates that constitute a useful setting for exploring
DNA nanodevices in physiological conditions.

4.5.2  DNA structures in serum

It is also important to study the stability of DNA structures in serum,
which is an important path for DNA structures to get to cells. Serum is
like lysates; it contains nucleases and lacking stabilizing salts such as
magnesium. Conway and colleagues once pointed out that small three-
stranded DNA nanostructures which in the shape of triangular prism were
more stable than individual strands in serum [49]. In the study, individual
strands had a half-life of less than an hour in 10% fetal bovine serum,
while the half-life of complete DNA structure was nearly two hours.
Furthermore, Perrault and colleagues studied that three different 3D DNA
origami in mammalian cell culture media with serum. Their results
b

showed that the stability of DNA origami in serum is related to the design
of DNA origami, the presence of Magnesium and the activity of nuclease
[50]. They observed that the samples of DNA origami with the addition of
6 mM magnesium did not denatured after one day incubation in that cell
culture media, whereas they denatured in the parallel experiment without
addition of magnesium. It is interesting that the sample of DNA-origami
nanotube is quite stable even without the addition of magnesium in cell
culture media. When more than 5% fetal bovine serum was added to
the media, all three 3D DNA structures were partially degraded by

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118 L. Wang and G. Arrabito

DNases; however, when adding actin (a protein that binds to nucleases) to

the system, nuclease degradation could be radically reduced. According to
other studies, it is shown that tetrahedral DNA nanostructures and DNA
origami are more stable than double-stranded DNA in the presence of
DNase [51, 52].
All the previous studies suggested that DNA nanostructures are more
stable than single/double stranded nucleic acids in physiological environ-
ment. Furthermore, some nanostructures can remain their structural integ-
.

rity in physiological conditions over long time period. It is worthwhile to

extend the research in understanding fully the interplay between the func-
tionality and stability of a DNA structure.

4.6  DNA Carriers for Drug Delivery

One of the most important applications of DNA nanostructures is the abil-
ity of these fancy structures to work as carriers for drug delivery. In this
part of the chapter, we will discuss the possible ways to involve DNA
nanostructures in drug delivery system.
The very first design of DNA-origami nanostructures working as
molecular containers for drug delivery was single-layer DNA origami.
Later on, they were further assembled into 3D nanostructures as shown in
Figures 4.7(a) and 4.7(b), a tetrahedron [53] and a hollow cube [54]. After
people realize the possibility of DNA structures working as drug delivery
carrier, various DNA nanostructures such as nanotubes [55, 56], cages
[57], and cubes [58] were designed and synthesized to demonstrate the
cellular uptakes of drugs both in vitro and in vivo. The successful delivery
of DNA nanostructures into cells creates new pathway for challenging
b

diverse therapeutic tasks. Jiang et al. [59] and Zhang et al. [60] demon-
strated a way to deliver doxorubicin (DOX) into cells in vivo by using
rod-like DNA origami or triangular origami as carriers (Figure 4.7(c)).
Usually, doxorubicin have low solubility and unfavorable side effects in
human body; however, these small drugs can hide behind the GC-rich
regions of DNA double helices, which gives great opportunity for DNA
nanostructures as new carriers to deliver them. It is worthwhile to mention
that; the work of Zhao and colleagues has demonstrated tunable DNA
nanostructures for optimal delivery of DOX to human breast cancer cells [61].

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DNA Nanostructures in Cell Biology and Medicine 119

.

Figure 4.7.    Potential DNA-based drug delivery vehicles and devices for triggering cell
signaling. (a) a tetrahedron [53] DNA-origami molecular containers. (b) a box with a
switchable lid DNA-origami molecular container [54], (c) DNA-origami nanostructures
for delivering DOX into cancer cells: an under-twisted rod-like shape [61], a straight rod
[59], and a triangle [59, 60]. (d) DNA structures for SiRNA delivery: a cage with cell-
targeting ligands (folate or peptide) at the end of the siRNA motifs [63]. (e) DNA struc-
tures for CpG-triggered immunostimulation: a DNA-origami tube [65] (f) A smart
logic-gated DNA-origami nanorobot to ­target cells and subsequently display the molecular
payload [69]. (g) Rectangular DNA origamis coated with virus CPs for efficient cellular
delivery: the complexes can adopt different conformations depending on the added CP
concentration [71]. (h) A virus-inspired membrane-encapsulated spherical DNA-origami
vehicle for decreasing immune activation and enhancing pharmacokinetic bioavailability
[46]. A tetrahedron was adapted with permission from Ref. [53]; Copyright (2009)
b

American Chemical Society. A box with a lid was adapted with permission from Ref. [54];
Copyright (2009) Nature Publishing Group. A straight rod and a triangle were adapted
with permission from Ref. [59]; Copyright (2012) American Chemical Society. An under-
twisted rod was adapted with permission from Ref. [61]; Copyright (2012) American
Chemical Society. siRNA cage was adapted with permission from Ref. [63]; Copyright
(2012) Nature Publishing Group. A CpG–DNA-origami tube was adapted with permission
from Ref. [65]; Copyright (2011) American Chemical Society. A nanorobot was adapted
with permission from Ref. [69]; Copyright (2012) The American Association for the
Advancement of Science. Rectangular DNA origamis coated with virus proteins were
adapted with permission from Ref. [71]; Copyright (2014) American Chemical Society.
A membrane-encapsulated origami was adapted with permission from Ref. [46];
Copyright (2014) American Chemical Society.

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120 L. Wang and G. Arrabito

In this work, they designed tunable DNA nanostructures that were able
to twist and control the release rate of the drug.
DNA nanostructures can be functionalized and then be used for
in vivo imaging [62]. In addition, DNA nanostructures can control
siRNA molecules for silencing genes. For example, Lee and colleagues
find that tetrahedral DNA nanostructures modified with folate or pep-
tide can load siRNAs, which can silence target genes in tumors [63]
(Figure 4.7(d)). In their research, they noticed that delivery of siRNAs by
.

DNA nanostructures strongly depends on the spatial orientation and the

density of the cancer-targeting ligands assembled on the carriers. After
few years, Shaw and colleagues demonstrate a way of using DNA-
origami “nanocapilars” to adjust the spatial orientation of the membrane-
binding ligands, which target to the membrane in cancer cells [64].
Those interesting findings showed the essence of DNA nanostructures of
being easily programmed and functionalized is very useful for working
as drug delivery carriers.
Afterwards, researchers led their interests in developing DNA-based
nanostructures for the application of programmable immunostimulants.
Schüller and colleagues successfully conjugate tens of cytosine–­
phosphate–guanine (CpG) sequences onto tubular DNA origami [65].
(Figure 4.7(e)) They found that using CpG decorated DNA origami
induced immunostimulation better than using lipofectamine (a common
transfection reagent) delivered CpG. Additionally, different from
Lipofectamine, CpG-origami are non-cytotoxic complexes. In 2015,
Sellner and colleagues introduced successful experiments with CpG-
coated origamis in vivo [56]. Moreover, several researches about different
DNA nanostructures as carriers for immunostimulatory CpG motifs were
b

successfully conducted, such as tetrahedral DNA cages [66], polypod-like

structured DNA [67] and DNA dendrimers [68]. All those successful
results give us a great hope of using DNA nanostructures as new drug car-
riers in cancer therapy. One of the most remarkable researches needs to be
point here is the logic-gate DNA-origami nanorobot developed by
Douglas et al. [69]. The nanorobot is a highly sophisticated delivery sys-
tem that can sense the surface proteins of the target cells and then trigger
programmed aptamer-based logic-gates thus selectively transport the
molecular payload to the cells (Figure 4.7(f)).

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DNA Nanostructures in Cell Biology and Medicine 121

Although all those great achievements in this field, we still need to

find an efficient transfection method for DNA nanostructure drug carriers.
Due to the reason that DNA origami are polar structures due to the back-
bone of nucleic acid, they open need modifications of peptide, cationic
polymer or lipid to successfully transport into cells. However, Brglez and
colleagues showed another possible way to improve the delivery effi-
ciency of DNA nanostructures. The surface properties of DNA origami
can be modified by the specific DNA intercalators thus subsequently
.

improve the transfection efficiency [70]. Furthermore, Mikkilä and col-

leagues decorated a single rectangular DNA-origami sheet with virus
capsid proteins (CPs) from cowpea chlorotic mottle virus (CCMV) [71].
The CP-origami complexes showed different morphologies, a roll or a flat
plate, which depends on the amount of CPs on DNA-origami (Figure
4.7(g)). It is noticed that the transfection rate increased significantly com-
pares to the bare DNA-origami structure of the experiments on human cell
line HEK293. Importantly, they didn’t observe any toxicity in their experi-
ments. Inspired by the viruses, Perrault and Shih [46] creatively encapsu-
late spherical DNA-origami structures in conjugated (PEGylated) lipid
membranes (Figure 4.7(h)). This invention smartly protects DNA-origami
nanostructures from nuclease digestion, and moreover, the immune acti-
vation in vivo experiments was significantly decreased.

4.7  Conclusion and Perspectives

Given the fundamental importance of DNA in biology, it is expected that
DNA nanotechnology could represent a real breakthrough in life sciences,
enabling a plethora of unprecedented, complex and exciting applications.
b

In this chapter, we showed that the extremely high flexibility in the design,
high cell permeability, biocompatibility and spatial positioning of self-
assembled DNA nanostructures permit them to be the ideal systems as
carriers for molecular payloads delivery, such as drugs, antibodies and
siRNA. We showed of ligands to be a powerful technique to mildly deco-
rate 2D or also 3D surfaces with capture DNA aiming at immobilizing
multiple proteins on the same surface or manipulate single cells by trig-
gering extracellular or intracellular responses. Future developments of
this approach, thanks to the integration of top-down nanofabrication

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122 L. Wang and G. Arrabito

approaches, will permit to immobilize gradients of ligands or mimicking

the topography and functionality of the extracellular matrix.
On the other hand, the field of DNA-origami has shown significant
development in the last decade and increasing numbers of labs and
researchers are now exploring such technology, with many potential excit-
ing applications being described. In particular, applications in drug
­delivery by DNA nanostructures can be implemented in practice by under-
standing of the pharmacokinetics and biodistribution immunostimulatory
.

properties of DNA nanostructures and finally by decreasing the produc-

tion costs, especially for large nanostructures such as DNA-origami.
Researches in this field will have to overcome the challenges associated
with the translation of these technologies to the clinic and widespread
biotechnology testing.

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Chapter 5
.

Targeting G-quadruplex DNA

as Potential Anti-cancer Therapy

Riccardo Bonsignore*,‡, Elisa Trippodo† and Giampaolo Barone†

*School of Chemistry, Cardiff University

Park Place, Cardiff, UK
†
Department of Biological, Chemical and Pharmaceutical
Sciences and Technologies University of Palermo
Viale delle Scienze, Ed. 17, 90128 Palermo, Italy
‡
BonsignoreR@cardiff.ac.uk

This chapter provides an introduction and also a review on non-canonical

DNA structures such as guanine-quadruplexes (G-quadruplexes) and their
b

targeting by small molecules for applications in Pharmacology. The arti-

cles considered in this chapter are mainly from 2016.

5.1 Introduction
When referring to the biological role of DNA in living beings it is easy to
relate it to the hard drive of a personal computer: as the latter, the polynu-
cleotide stores all the information needed for a proper cellular life. Any
molecule capable to interfere with the DNA, blocking its function, may

129

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130 R. Bonsignore, E. Trippodo and G. Barone

therefore become lethal for a cell. For such a reason the polynucleotide
has been widely considered an important target to halt cell proliferation,
especially in life-threating diseases as cancer. Nonetheless, DNA-binders,
being unable to discriminate between the DNA of healthy and cancerous
cells, have been always characterized by several side effects. This,
together with the recent finding of more specific targets, e.g. proteins,
shifted the interests of the researchers towards the latter.
Despite this, recent discoveries about the involvement of non-­
.

canonical DNA secondary structures in several pathologies — cancer,

above all — have again turned on the lights on the polynucleotide as a
potential target in anti-cancer therapies. The hypothesis is that, when a
molecule is capable to interact with a specific secondary structure of the
polynucleotide, different from the canonical double helix, it could affect
mainly cancerous cells causing fewer side effects. Among these struc-
tures, G-quadruplexes sequences have been widely found to be involved
in cancer progression and therefore are nowadays considered as potential
targets of small binders.
In the present chapter we intended to focus our attentions on the
advances in G-quadruplex DNA targeting by small molecules in the year
2016, mainly in an anti-cancer view. For such a reason, the first two para-
graphs will give basic information about the structural and biological
aspects of G-quadruplex DNA. These themes have been widely and prop-
erly discussed in several reviews and books, which we address the readers
to refer to for wider and more detailed information [1–3]. In the second
part of the chapter we will introduce the reader to recent discoveries of
G-quadruplex DNA binders, discussing typical design processes.
b

5.2  Structural Details

The propensity of guanine-enriched nucleic acids to self-associate has
been recognized since 1910 when Bang showed these sequences could
form gel-like substances in water [4]. This evidence was later supported
by spectroscopic studies that allowed Ralph et al. [5], to realize that the
optical density of tri- and tetradeoxyguanosines changed with tempera-
ture. In particular a thermal transition point was evident, pointing out the
existence of an ordered secondary structure. Nonetheless, the real turning

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 131

point in the understanding the nature of G-quadruplex DNA was found

still in the 1962 when Gellert et al., through X-ray diffraction studies on
gels of 3′- and 5′-guanosine monophosphate, demonstrated that their
arrangement was similar to that of double-helical DNA [6]. Notably,
according to the authors, the high stability of this secondary nucleotide
structure could be only explained by the hydrogen-bonding of four gua-
nine bases.
The building block of a G-quadruplex DNA is indeed made up of a
.

tetrad of guanines linked by Hoogsteen hydrogen bonds involving the N1,

N2, N7 and O6 atoms of each guanine base (Figure 5.1(a)). The final
quadruplex structure is stabilized by π–π interactions among the adjacent
stacked tetrads (Figure 5.1(b)), whose misalignment (Figure 5.1(c)) gives
rise to a quadruple helical-like structure. Notably, the formation of these
square planar platforms is driven by the presence of a monovalent cation,
typically K+ or Na+, equidistant from the eight O6 atoms of two adjacent
tetrads [7, 8], and forming a ionic channel in the center of the whole
G-quadruplex (Figure 5.1(c)).
As double-helical DNA, also G-quadruplex DNA presents a high
polymorphic nature: as a matter of fact, these structures can easily adopt
different conformations depending on the solution conditions and on the
nature of monovalent ion. In this respect, due to the higher intracellular
concentration of K+ than Na+ (about 140 mM vs. 10 mM), the potassium
driven structure is considered to be biologically more relevant [8].
Moreover, each of the guanine residues can adopt either a syn- or an anti-
glycosidic conformation, giving rise to 16 possible conformations per
tetrad and many more when considering a whole G-quadruplex [9].
G-quadruplex DNA can arise from the folding of a single (intramo-
b

lecular) guanine enriched G-tract, or from two up to four guanine-enriched

separate strands, forming an intermolecular G-quadruplex [3]. Moreover,
according to the orientations of the strands, G-quadruplexes can be clas-
sified as parallel, antiparallel and mixed, or hybrid, with several possible
connecting tracts (loops), thus increasing their polymorphism.
From the considerations above it becomes clear that the knowledge of
the in vivo structure of the G-quadruplex DNA is an important challenge
in order to design optimal molecules with enhanced biological properties,
capable to bind these structure for therapeutic purposes, as it will be

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132 R. Bonsignore, E. Trippodo and G. Barone

.
b

Figure 5.1.    (a) Representation of a G-tetrad with Hoogsteen hydrogen bonds; (b) front
and (c) top view of a G-quadruplex from c-KIT DNA (Adapted with permission from A.T.
Phan et al. J. Am. Chem. Soc., 2007, 129, 4386–4392. Copyright (2007) American
Chemical Society); (d) in vivo visualization of G-quadruplex DNA (red dots) by means of
a fluorescent antibody. Adapted with permission from Ref. [11]. Copyright (2013) Nature
Publishing Group.

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 133

discussed in the following paragraphs. In such a context, X-ray crystal-

lography and NMR spectroscopy have been widely and predominantly
used to characterize structural aspects of G-quadruplex DNA.

5.3  Biological Involvement

More than 30 years ago, Aaron Klug stated that since G-quadruplexes
could form so easily in vitro, Nature should have found a way to use them
.

in Biology. This statement postulated the involvement of these structures

in complex biological functions, despite their observation in vivo was not
yet achieved. The development of specific antibodies directed against
G-quadruplex folded DNA represented the real turning point to confirm
their existence and to localize them in living cells. For example, by in situ
immuno-staining, Schaffitzel and coworkers in the 2001 were able to
localize and visualize the telomeric G-quadruplex DNA in the micronu-
clei of the ciliate Stylonychia lemnae [10]. As for human DNA, the direct
in vivo visualization of the G-quadruplex DNA was recently achieved by
Balasubramanian’s group in 2013 by means of a specific engineered anti-
body [11]. As shown in Figure 5.1(d), stable G-quadruplex motifs were
found in telomeres, gene bodies and gene regulatory regions [12].
Interestingly, bioinformatic analysis of the human genome previously
revealed that over 300,000 human DNA sequences might fold into a
G-quadruplex [13]: these sequences are not-randomly distributed but
mainly located in telomeric regions and in proximity of gene promoters.
In particular, the development of next-generation sequencing and poly-
merase stop assays have allowed the identification of more than 700,000
human G-quadruplexes, of which almost 450,000 have not been predicted
b

before, such as BRCA1 and BRCA2 genes [2]. Overall, these findings
point out the regulatory role played by G-quadruplex DNA, in the tran-
scription of genes and in the replication of human genome, as it will be
discussed in the next paragraphs.

5.3.1  G-quadruplexes in human oncogene promoters

The first evidences of “altered DNA conformations” in the nuclease hyper-
sensitivity of promoter elements were found in 1982, when Larsen et al.
investigated the chicken β-globulin gene [14]. Later, several G-enriched

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134 R. Bonsignore, E. Trippodo and G. Barone

promoter sequences, potentially able to fold into G-quadruplexes, were

identified in a wide amount of human genes involved in growth and pro-
liferation processes, including many of the human oncogenes [15]. For
example, the occurrence of these structures has been found in genes such
as human Vascular Endothelial Growth Factor (VEGF) [16], c-MYC
[17, 18], Hypoxia-Inducible Factor-1α (HIF-1α) [19], B-Cell Lymphoma-2
(BCL-2) [20–22], sMtCK [23], human and mouse Kirsten RAt Sarcoma
(KRAS) [24], human c-KIT [25, 26], REarranged during Transfection
.

(RET) [27] and Platelet-Derived Growth Factor-A (PDGF-A) [28].

Among all of them, the c-MYC oncogene was the first human gene
systematically studied as a G-quadruplex target for anti-cancer ther-
­
apy, due to its importance in cancer proliferation and also because its
protein, MYC, is quite unstable and therefore hard to be targeted by any
drug [1].
These discoveries raised the interest of several scientists, since the
stabilization of a G-quadruplex motif in promoter oncogenes could allow
the down regulation of genes playing key roles in cancer progression. The
assumption is that, when a small molecule binds to a G-quadruplex folded
DNA sequence, RNA polymerase and transcription factors, as well, are
not anymore able to recognize these DNA sequences: as a consequence a
mRNA will not be produced and the amount of the related-synthesized
proteins will decrease. Furthermore, this effect would be ideal when the
binding ligand is capable of selectively recognizing the targeted
G-quadruplex of the overexpressed gene sequence, rather than any other
G-quadruplex or duplex DNA. Nonetheless, such specificity still remains
one of the major challenges and main goals of the “G-quadruplex target-
ing” research.
b

5.3.2  G-quadruplexes in telomeres

Human telomeres, at the terminus of chromosomes, are characterized by
single-strand overhangs of repeated TTAGGG sequences. Telomeric DNA
physiologically shortens during each replication cycle as a consequence of
the inability of the DNA-replication machinery of the cell to fully repli-
cate these ends [29]. Therefore, in the absence of compensating mecha-
nisms, telomeres in normal cells progressively shorten until they reach the
limit point — called Hayflick limit — when cells respond by ceasing

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replication and entering senesce, normally leading to apoptosis [30].

Nevertheless, some cells, such as stem and germ cells, have the need to
elongate the telomeres ends, thus keeping their unlimited proliferative
capabilities. In particular, for this purpose, these cells synthesize an
enzyme named telomerase, owning reverse transcriptase activity. This
enzyme is not expressed in other healthy cells, causing a loss of 50–200
bases at the telomeric region per replication cycle. Interestingly, telomer-
ase has been also found to be expressed in most of human cancers (85–
.

90%) [31] granting them unlimited proliferation ability and consequent

immortalization. On the other hand, the occurrence of G-quadruplex sec-
ondary structures in these G-rich tracts allows designing specific
G-quadruplex binders and stabilizers: once stabilized, indeed, telomerase
should not be anymore able to recognize its natural substrate and therefore
no elongation of the telomeres would take place. As a consequence, can-
cer cells might enter senescence and probably undergo apoptosis as a
healthy human cell. However, the employment of telomerase inhibitors is
limited by the presence of alternative lengthening mechanisms (ALT) in
cancer cells, which maintain the integrity of telomeres and, consequently,
their unlimited replicative ability [32]. Notably, recent findings seem to
point out that the interaction between stabilizing molecules and telomeric
G-quadruplexes might bring to the uncapping of telomeres from constitu-
tive proteins, damaging these ends. In such a context it seems that the
inhibition of the telomerase activity might play a secondary but still pre-
cious role [2].
It is worth noticing that the same specificity-issues reported in Section
5.3.1 are also related to the design and synthesis of telomeric G-quadruplex
binders, thus pushing scientists to exploit structure-activity relationships
b

to achieve a specific interaction with the aimed G-quadruplex, goal yet

still far to be reached.

5.4 Recent Advances in Targeting

G-quadruplex DNA
A multidisciplinary approach is nowadays used to assess the DNA-
binding properties of G-quadruplex stabilizers: among the experimental
techniques exploited, those such as UV–Vis, Circular Dichroism (CD),
NMR spectroscopies, as well as ESI–MS and FRET melting assays, are

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136 R. Bonsignore, E. Trippodo and G. Barone

HN

O O N
N N N NH N N
H H N
O N N
F
N O O
.

(1) (2)

Figure 5.2.    Molecular structures of BRACO-19 (1) and Quarfloxin (2).

commonly used to collect clues of the G-quadruplex binding capabilities

of newly synthesized compounds. These results are often corroborated by
computational investigations, through molecular docking and/or molecu-
lar dynamics (MD) simulations, as well as by biological studies in vitro,
such as cell cycle analysis, Western Blotting of the expression of the gene,
telomerase activity and antiproliferative assays.
Noteworthy, among the newly synthesized molecules with
G-quadruplex stabilizing abilities, the discoveries of compounds such as
BRACO-19 (1) [33] and Quarfloxin (2) [34] represent a milestone to
guide the design of new molecules (see Figure 5.2).
Since then, indeed, researchers have been exploring the suitable fea-
tures that a compound must possess in order to preferentially bind
G-quadruplex rather than double-helical DNA. One of the widest explored
binding modes is the top-stacking of a molecule on one of the two exter-
nal faces (3′ or 5′) of the G-quadruplex motifs. In such a context, two main
b

structural features are needed for a molecule to establish this kind of

interaction:

(i) a flat planar π-system, allowing π-π interactions between the binder
and the top quartet of the G-quadruplex;
(ii) the presence of polar side chains, possibly positively charged at physi-
ologic pH, able to interact with the negatively charged sugar–­
phosphate backbone in the DNA grooves thus reinforcing the
interaction with the G-quadruplex.

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 137

On the other hand, some compounds have been designed as

G-quadruplex groove binders. The latter feature has been exploited to
induce a certain degree of selectivity among G-quadruplex sequences, and
to increase the affinity for particular structural properties possessed by
certain G-quadruplex conformations. For example, the NMR structure of
G-quadruplex DNA from c-KIT sequence has shown to possess a deep
hydrophobic cleft never found in other G-quadruplexes or in duplex DNA.
Therefore, designing a molecule capable to structurally fit with such
.

groove might reveal to be a powerful strategy to achieve a binding speci-

ficity that could reduce side effects in anti-cancer therapies.
For the same reason, many research groups have reported the increase
in G-quadruplex binding capabilities of molecules in which a metal ion is
complexed by a suitable organic ligand. In fact, the presence of a cationic
metal ion usually increases water solubility, which is necessary since the
final interaction occurs in cell environments, where water is the solvent.
Moreover, the presence of a metal ion allows a fine-tuning of the struc-
tural properties of the complex, which can be exploited in order to
increase the interaction with the grooves or with the guanine tetrads. In the
latter case, it is presumable that the presence of a metal ion drives the
positioning on the top face of the G-quadruplex, by aligning itself on top
of the internal K+ or Na+ channel.
For such a reason the following chapter is dedicated to the literature
findings of the year 2016, concerning organic ligands and metal-complexes
as G-quadruplex binders.

5.4.1  Targeting G-quadruplex DNA with

b

organic binders
Small isoquinoline alkaloids, found in several plants, have been widely
used by traditional medicine for the treatment of several diseases, from
fever to cancer [35, 36]. Among them, derivative 4 has been found to pref-
erentially bind human G-quadruplex DNA [37–40]. In detail, Malhotra and
coworkers, after setting up a convenient route to synthesize dihydrocheler-
ythrine (3), were able to assess its G-quadruplex binding capabilities
after a comparison with the parental alkaloid 4, using biophysical and

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138 R. Bonsignore, E. Trippodo and G. Barone

biochemical approaches [41]. In particular, ligand induced thermal stabili-

zation experiments through CD spectroscopy (CD melting experiments)
were carried out in order to unravel the stabilizing capabilities of 3 and 4
towards c-MYC and c-KIT1 G-quadruplexes. When the DNA secondary
structure is stabilized by means of a binder it is in fact expected that the
amount of the energy needed to separate its filaments increases with
respect to the polynucleotide alone: thus an increase of the melting tem-
perature of the DNA occurs. Malhotra et al. found that, on c-MYC
.

G-quadruplex, 3 caused a larger stabilization than 4, and that this order was
reversed for c-KIT1 G-quadruplex. Nonetheless, no interaction with
duplex DNA was found, since its melting temperature remained constant
upon addition of both derivatives. Computational investigations on the
binding mode of 3 to the c-MYC G-quadruplex revealed π-stacking to both
the top both the bottom quartets of the G-quadruplex as the main binding
mode. Lastly, biological assays showed that 3 exhibited a 30% inhibitory
effect on the proliferation of the only A431 cancer cell line. This result
pointed out interesting cancer-tissue specificity for this derivative [41].
Similar c-MYC selectivity, over duplex DNA and other
G-quadruplexes, was found by Kumar and coworkers in the early 2016.
The authors, indeed, employing one-pot modular click reactions were
capable to synthesize two dansyl-guanosine conjugates, with the deriva-
tive 5 showing the most interesting G-quadruplex binding properties [42]
(see Figure 5.3). NMR spectroscopic titrations of c-MYC G-quadruplex
in the presence of increasing amounts of 5 revealed line broadening and
chemical-shift perturbations of the NMR spectra of the G-quadruplex
alone. This result allowed the authors to suggest that 5 preferentially binds
to 3′-end-capping structure and to the groove region of the G-quadruplex.
b

Moreover, antiproliferative assays were carried out on HT1080 cell lines,

which are characterized by an overexpression of c-MYC oncogene. These
studies showed that compound 5 could cause a dose-dependent inhibition
in cell proliferation, thus addressing its in vitro anticancer properties.
Noteworthy, by means of other biological approaches — such as quantita-
tive real-time PCR (qRT-PCR) — Kumar et al. were able to demonstrate
a gradual decrease, in the presence of increasing concentration of 5, of the
amount of c-MYC transcript. Despite the exact mechanism underlying
this repression has not yet been found, this result can be considered

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 139

.

Figure 5.3.    Molecular structures of compounds 3–5.

consistent with the deactivation of c-MYC promoter, through the stabili-

zation of its G-quadruplex by 5.
A specific interaction with parallel G-quadruplex DNA was found
by Diveshkumar et al. when testing compounds 6 and 7 [43] (see
Figure 5.4). As depicted in Figure 5.5, both of them are indolylmethyle-
neindanone-based molecules presenting a positive charge, which, above
all, increases their water solubility. The authors, through CD melting
experiments, assessed their higher stabilization of the parallel c-MYC and
b

c-KIT G-quadruplexes with respect to both the hybrid telomeric

G-quadruplex and duplex DNA. These results were corroborated by sev-
eral studies such as ESI–MS experiments. The latter are commonly used
to assess the occurrence of non-covalent interactions between a
G-quadruplex and a small binding molecule and allow determining the
affinity constant for the resulting complex [44, 45]. It was therefore found
that derivative 6 was a stronger stabilizer of both c-MYC and c-KIT
G-quadruplexes than 7, probably for the presence of the ethyl instead of
the propyl side chains of 7 [43]. Notably, by employing MD simulations,

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140 R. Bonsignore, E. Trippodo and G. Barone

.

Figure 5.4.    MD simulations of 6 with (a) c-MYC G-quadruplex; (b) 5′-quartet and (c, d)
3′-quartet of the G-quadruplex. Adapted with permission from Ref. [43]. Copyright (2016)
American Chemical Society.
b

the authors revealed that the main stabilizing interaction was π-stacking of
the two aromatic groups, indole and indanone, with the top and bottom
G-quartets and with the flanking bases of c-MYC and c-KIT G-quadruplexes.
This π-stacking was not observed with telomeric G-quadruplex DNA,
thus addressing the specific interaction of 6 and 7 with c-MYC and c-KIT
rather than with telomeric G-quadruplex DNA to their different topolo-
gies. This last evidence was even pointed out by Taq polymerase stop
assays, where both molecules showed a low and a high IC50 value for
c-MYC template and for the telomeric sequence, respectively [43].

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 141

Interestingly, other indole-based molecules have been reported

in 2016 as promising G-quadruplex stabilizers. As an example, Livendahl
et al. synthesized several 2,2′-diindolylmethanes derivatives and tested
them as G-quadruplex binders by means of spectroscopic and biologic
investigations [46]. Among all of the synthesized molecules, 9(a), 9(b)
and 10 in Figure 5.5 have shown the best G-quadruplex binding capabili-
ties, especially when compared to the well-known G-quadruplex stabilizer
Phen-DC3, 8. Notably, even these authors found their compounds able to
.

bind more efficiently to certain G-quadruplex sequences, thus allowing

them to conclude that upcoming chemical modification of these molecules
might enhance their binding selectivity [46].
Moreover, Amato and coworkers recently reported the synthesis of
several hydrazone-based compounds as G-quadruplex binders. Among
them, derivative 11, 12 and 13 showed a preferential binding to parallel
topologies over human telomeric and duplex DNA, as obtained through
CD melting assays [47] (see Figure 5.6).
Molecular docking approaches suggested that the tricyclic
diimidazo[1,2-a:1,2-c]pyrimidine core of 12 could easily fill the spaces
both in the top and in the bottom faces of c-MYC G-quadruplex. Notably,
additional hydrogen bonds and electrostatic attraction reinforced the inter-
actions of 12 with the selected G-quadruplex. Remarkably, the molecule
was even found to be the more effective in inhibiting human U2OS and
HCT116 cancer cell growth.
Pradeepkumar and coworkers have found a similar binding specificity
toward parallel G-quadruplex topologies. Having synthesized three
benzothiazole hydrazone of furylbenzamides (14(a), 14(b) and 14(c) in
Figure 5.7) with different side chains, the authors assessed their
b

G-quadruplex stabilization properties by means of fluorescence and CD

melting assays [48]. Interestingly, all of them showed a strong stabilization
of parallel G-quadruplex DNA topologies such as those of c-MYC and
c-KIT1; on the contrary, the three derivatives presented weaker interactions
with antiparallel (h-RAS1) and hybrid (telomeric) G-quadruplexes, as well
as with duplex DNA. Notably, derivative 14(b), which from the biophysical
assays has been recognized as the best of the series, could also inhibit the
Taq DNA polymerase of c-MYC template in a micromolar concentration;
this value, being about 200-fold lower than the one needed for the telom-
eric template, confirmed the preference of interaction with parallel

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142 R. Bonsignore, E. Trippodo and G. Barone

.

9(a) 9(b)
b

Figure 5.5.    Molecular structures of compounds 6–10.

G-quadruplex topologies. Lastly, computational approaches, used to collect

clues about the biding modes of the molecules to c-MYC G-quadruplex
DNA, allowed assessing a 1:1 binding stoichiometry in which π-stacking
and electrostatic interactions were the main stabilizing forces.

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 143

.
b

Figure 5.6.  CD melting curves of c-KIT (left column) and c-MYC (right col-
umn) G-quadruplex DNA in presence of increasing amounts of 11 (a, b), 12 (c, d) and 13
(e, f). Adapted with permission from Ref. [47]. Copyright (2016) American Chemical
Society.

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144 R. Bonsignore, E. Trippodo and G. Barone

.

14(a) 14(b) 14(c)

Figure 5.7.    Molecular structures of compounds 11–14.

In the search for c-MYC G-quadruplex selective binders, Jiang and

coworkers have reported on the synthesis of novel 4-anilinoquinazoline
derivatives [49]. Biophysical studies allowed them to assess that the pres-
ence of an aniline group in the quinozaline scaffold, together with two
positive charges electrostatically bonding to the loops and groves of the
G-quadruplex, improved the binding capabilities of the synthesized mol-
ecules, without affecting the duplex DNA structure. Notably, the authors
carried out FRET titrations, which allowed them selecting molecule 15 in
Figure 5.8 as the most promising derivative of the series. Indeed, in order
to carry out a FRET melting assays, a polynucleotide is labeled with a
fluorophore on one of its ends and a fluorescence quencher on the oppo-
b

site end. Their proximity allows quenching the fluorescence of the labeled
polynucleotide. On the contrary, when temperature is increased the dena-
turation of the DNA occurs leading to the separation of the bases and
therefore moving apart the fluorophore and the quencher. For such a rea-
son, an increase of the fluorescence signal can be recorded and allows
determining the temperature of melting (Tm) of the polynucleotide. Once
a molecule is stabilizing the duplex or the G-quadruplex DNA, more
energy is needed to break the hydrogen bonds between the bases and
therefore a shift of the Tm toward higher temperature is expected.

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.

Figure 5.8.    Molecular structures of compounds 15–20.

b

In particular, derivative 15 has shown an about 20°C increase in the Tm of

the c-MYC G-quadruplex DNA, pointing out its strong binding to the lat-
ter. The authors have recognized this stabilization as the most probably
cause for the down-regulation of the expression of the protein c-MYC
when HeLa cells were exposed to 15. Further investigations have even
proved that the molecule did not affect the growth of normal primary

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146 R. Bonsignore, E. Trippodo and G. Barone

cultured mouse mesangial cells, despite having an antiproliferative effect

on HeLa cell lines, with their arrest in G0/G1 cell cycle phase.
Micheli et al. has recently deepened the studies on derivative 16
(Figure 5.8) [50] — addressed in a past work as a human telomeric
G-quadruplex stabilizer [51] — investigating on its ability to stabilize
also c-MYC and BCL-2 G-quadruplex DNA. The molecule is structur-
ally related to both compounds 17 and 18, which for such a reason were
included in the assays carried out. Collectively, biophysical experiments
.

such as ESI–MS, FRET and CD titrations have shown 16 and 17 have

comparable G-quadruplex stabilizing capabilities, outperforming the
other related molecule, 18. As already mentioned for several compounds
reported in this paragraph, authors have shown that 16 could interact
with G-quadruplex DNA through π-stacking to the ending G-quartets,
even though no selectivity of interaction with the c-MYC and BCL-2
could be observed. Lastly, qRT-PCR and western blotting analysis
showed that 16 was the most effective molecule in reducing the protein
levels of both of the genes. Consequently, the authors concluded that the
anti-cancer capabilities of the derivative could not be merely related to
its interaction with human telomeric G-quadruplex DNA but also with
other G-quadruplexes. Notably, since 16 has shown to be a better in vitro
antiproliferative derivative than compounds 17 and 18, it was considered
a suitable candidate for developing novel and more specific G-quadruplex
binders.
Lastly, the anti-cancer capabilities of anthracyclines have been widely
known to be related to a multiple mechanism of action, which can, directly
or indirectly, even involve the duplex DNA [52]. Nemorubicin and doxo-
rubicin, respectively 19 and 20 in Figure 5.7, are indeed known to be
b

DNA-intercalators. Nevertheless, their molecular structures meet almost

all the requirements to interact also with G-quadruplex DNA. For such a
reason, Scaglioni and coworkers performed NMR and ESI–MS experi-
ments with an oligonucleotide containing the TTAGGGT repeated
sequence, typical of the human telomeres, in order to assess the
G-quadruplex binding capabilities of both 19 and 20 [52]. Not surprisingly,
the authors found that similar interactions with the telomeric G-quadruplex
DNA could be achieved in presence of the two molecules and structural
evidences of the interactions with the polynucleotide could be collected
from their experiments. In particular 19, which showed the best binding

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 147

capabilities, has been found to be able to intercalate between the A3 of the

loop and the G-tetrad, in the 5′-end of the G-quadruplex; at the same time,
the molecule was also capable to intercalate between the G6 and the T6,
in the 3′-end. A similar 2:1 stoichiometry of interaction was also found
when the authors tested 19 with c-MYC sequence, by means of the same
spectroscopic approaches. Nonetheless, they pointed out in the latter case
that the interaction with the 5′-end should be much more favorable, because
seven π-interactions could be concomitantly achieved. In conclusion, the
.

findings of the authors clearly demonstrated the capabilities of 19 and 20

to interact also with G-quadruplex DNA therefore allowing them to infer
that their anti-cancer capabilities could be also due to these interactions.

5.4.2  Targeting G-quadruplex DNA with

metal complexes
Ruthenium based molecules have been widely investigated in past dec-
ades as promising anti-cancer candidates: indeed, displaying a remarkable
in vitro anti-cancer activity, this metal has gained a role as a possible
alternative to platinum based drugs [53]. Notably, complexes such as
NAMI A and KP1019 (21 and 22 in Figure 5.9) have also undergone
phase I and II clinical trials [54–56].
He et al., in 2016 synthesized three octahedral RuII complexes with
aromatic planar ligands: among them, complex 24 (Figure 5.9) bearing two
ammonia groups along the two z-axial position, showed the best DNA
binding capabilities [57]. By means of biophysical assays, the authors
proved the occurrence of significant topological selectivity of 24 towards
both the antiparallel telomeric G-quadruplexes investigated (hybrid and
b

basket-type) rather than towards the parallel conformation, represented by

c-MYC. The thermal stability and the CD spectra of c-MYC G-quadruplex
were, indeed, much less altered in presence of the derivative. Moreover, the
comparison between the two antiparallel telomeric G-quadruplexs binding
with 24, indicated a higher structural fitting with the basket-type rather
than with the hybrid telomeric G-quadruplex. Computational studies con-
firmed the occurrence of π-stacking of 24 with the G-quartets. Notably, the
presence of the ammonia groups allowed achieving unique extra stabiliz-
ing effects: in fact NH3 could insert into the central cationic channel and
interact with the bases through hydrogen bonds.

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148 R. Bonsignore, E. Trippodo and G. Barone

.

Figure 5.9.    Molecular structures of compounds 21–25.

Moreover, complex 24 exhibited an efficient inhibition of the telom-

erase activity, at a much lower concentration than that needed for
BRACO-19 (1) and TMPyP4 (23) [58], well-known G-quadruplex bind-
ers. Lastly, its cytotoxicity was investigated against HeLa, MDA–MB231,
HT 1080 and MCF-7 cancer cell lines, showing that 24 was about 2 times
b

more potent than TMPyP4. Remarkably, such RuII complex was even less
cytotoxic towards normal cells [57].
Another interesting study has been carried out by Wu and coworkers,
synthesizing a series of arene Ru complexes with different halides substi-
tuting an hydrogen atom, and studying the influence of this substituent on
c-MYC G-quadruplex binding and its biological consequences [59].
Results, supported by computational studies, have demonstrated that all
complexes are able to bind to the grooves of the G-quadruplex but
­derivative 25 in Figure 5.9, bearing a chloride as substituent, was the best

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 149

of the series, showing a higher affinity toward the G-quadruplex in the

biophysical assays. Indeed, atomic radii seemed to play a key role in the
groove-binding process. In particular, CD titrations showed remarkable
variations on c-MYC spectra in presence of increasing amounts of 25,
which are consistent with conformational changes occurring to
G-quadruplex DNA’s secondary structure.
Wu et al. also used the FITC-gelatin invasion assay to assess the
­anti-cancer capabilities of the arene–Ru complexes. Indeed cells with high
.

invasion rates can form protrusion of the plasma membrane (namely inva-
dopodia) and release matrix metalloproteinases. The latter degrade the
fluorescent gelatin and therefore the appearance of dark holes or of non-
fluorescent areas occurs. MDA–MB231 cells, without treatment with
complex 25, formed lots of black holes in FITC-gelatin, whereas after
their exposure to 25 the dark areas decreased (Figure 5.10), proving a
marked inhibition of invasion of MDA–MB231 cells [59].
The toxicity of the molecule has been tested using zebrafish embryos.
It is worth reminding that zebrafish DNA possesses a high homology with
human DNA and therefore they are considered reliable models for human
b

Figure 5.10.  FITC gelatin assays of derivative 25. Adapted with permission from
Ref. [59]. Copyright (2016) American Chemical Society.

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150 R. Bonsignore, E. Trippodo and G. Barone

S O O
N
N
N N N
Ru Me NH HN Me
N N
N
N N N
N

26 27

Me
.

N I
Me Pt NH2 NH
N I N I
Pt NH2 R
N I O O
N
Me NH HN Me
N N

28 29

Figure 5.11.    Molecular structures of compounds 26–29.

DNA-binders. All the zebrafish embryos, without treatment with complex

25, developed into juvenile zebrafish; interestingly the development of the
embryos was even achieved after exposure to increasing concentrations of
25, suggesting its low toxicity for the zebrafish embryos [59].
Interestingly, Wang and coworkers synthesized and characterized
two similar ruthenium(II) complexes, an example of which is 26 in
Figure 5.11 [60]. By means of biophysical assays they proved that both
complexes were able to strongly bind and stabilize human telomeric
b

G-quadruplex DNA. In particular, Wang et al. carried out a Fluorescent

Intercalator Displacement (FID) assay, based on the competitive binding
with DNA by synthetic molecules and Thiazole Orange (TO). Indeed, TO,
an organic molecule with a high affinity towards DNA, displays a fluores-
cence signal when it is bound to the polynucleotide; on the contrary in pres-
ence of a competitive ligand for G-quadruplex DNA, its displacement occurs
and therefore a loss of the fluorescence of TO is achieved.
The two RuII complexes were able to displace TO at low concentra-
tions, resulting stronger G-quadruplex binders than TO. Moreover both of

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 151

them, but in particular 26, displayed a preferential binding to human telo-

meric G-quadruplex rather than to duplex DNA [60].
Platinum complexes have been widely studied as anti-cancer agent
since the discovery of cisplatin. This complex, able to bind covalently
B-DNA, is still used in anti-cancer treatments and, for this reason, is often
included in binding assays as a comparison.
Betzer and coworkers conjugated the N-heterocyclic carbene plati-
num complex 27, which had already showed anti-tumor properties, with
.

the G-quadruplex binder PDC 28 [61]. The synthesized complex, 29,

displayed enhanced properties with respect to 27 and 28 alone, with a
binding process resulting from the cooperation of the two components of
the molecule. Indeed, 29 was able to bind preferentially and irreversibly
human telomeric G-quadruplex sequences with a stronger affinity than 28.
Moreover, by means of biophysical assays Betzer et al. proved that 29
bound covalently G-quadruplex DNA with a different platination process.
By isolating platination products it was possible to establish the metallic
coordination of 29 with adenines A7 and A19 differently from the com-
plex 27 that was coordinated with adenines A7 and A13. This result
allowed deducing that the binding of 29 was driven by the stacking of the
PDC moiety 28 onto the G4-structure. Moreover, biological assays proved
that the conjugated complex 29 induced an increased loss of TRF2, a telo-
meric protein essential for the maintenance of telomeres [62], compared
to 27 and 28 alone [61].
Furthermore, Gama and coworkers synthesized a series of a­ nthracene–
terpyridine Pt(II) and Cu(II) complexes containing different linkers [63].
By biophysical assays these derivatives showed affinity and selectivity for
human telomeric and c-MYC G-quadruplexes over duplex DNA. Notably,
b

the metal-free ligands did not display any binding capabilities towards
duplex and G-quadruplex DNA, highlighting the importance of the metal-
lic center. Interestingly, the thermal stabilization of the G-quadruplexes
induced by these compounds was higher in the case of platinum(II) com-
plexes than of the copper(II) ones. Furthermore, complexes with a longer
linker, for example 30, showed enhanced interaction with G-quadruplex
forming sequences [63].
Moreover, Duskova and coworkers studied the interaction of a series
of Cu(II) complexes of carbohydrate ligands, whose general structure, 31,

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152 R. Bonsignore, E. Trippodo and G. Barone

O N Pt Cl

O N
HN
.

30

O O
H H O O H2O
N N N N
R R N N
Cu Cu
N N
N N O
H2O O
O O

R = sugar
31 32

Figure 5.12.    Molecular structures of compounds 30–32.

is reported in Figure 5.12, with human telomeric G-quadruplex DNA [64].

Interestingly, among the complexes containing carbohydrate units of the
D-series, the glucose and mannose derivatives were the most able to dis-
place TO in the G-quadruplex FID assay and showed higher selectivity for
G-quadruplex over duplex DNA under potassium ionic conditions. On the
contrary, for the L-series containing complexes the authors found the best
derivative in the rhamnose complex, possessing the higher binding affinity
and selectivity. It also exhibited the most drastic modification of the typi-
b

cal CD spectra of G-quadruplex DNA, suggesting the occurrence of a

considerable modification of the secondary structure of the polynucleo-
tide. Interestingly, this rhamnose derivate outperformed all of the other
Cu(II) complexes. In conclusion, the authors were able to suppose an
interaction mode of the synthesized compounds which involves their
binding with the loops and grooves of the polynucleotide, despite
­
their π-stacking, as supported by TO displacement, still plays an important
role [64].
Concerning other copper(II) complexes, Housaindokht reported about
a novel compound, 32, and, by means of biophysical assays, studied its

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 153

G-quadruplex binding capabilities [65]. The UV–Vis spectra of the com-

plex, recorded in presence of increasing amounts of the polynucleotide,
showed a hypochromic effect, typical of the interaction with DNA.
Moreover, MD simulations confirmed the ability of 32 to stack on the
quartets and stabilize the structure. Thermodynamic parameters calculated
from the biophysical assays suggested that the binding process was driven
by the entropy and displayed the key role of hydrophobic forces in the
interaction of 32 with G-quadruplex DNA [65]. Moreover the negative
.

values of ΔG indicated the spontaneity of the binding process.

Lastly, for their extended π-system, Schiff base derivatives such as
Salen-like and Salphen-like transition metal complexes have been widely
investigated for their DNA-binding capabilities. A series of publications
has indeed pointed out that these molecules act as DNA-intercalators and
as G-quadruplex stabilizers [66–70]. In such a context, Bonsignore et al.
reported the synthesis of two novel zinc(II) and nickel(II) Salphen-like
compounds of which the nickel(II) complex, 33 in Figure 5.13, showed to
be the only one capable to interact with telomeric G-quadruplex [71]. By
combining UV–Vis and CD spectroscopies, the authors assessed the pref-
erential binding of this derivative with human telomeric G-quadruplex
b

Figure 5.13.    Molecular structures of compounds 33–35.

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154 R. Bonsignore, E. Trippodo and G. Barone

with respect to duplex DNA. Notably, MD simulations pointed out that 33

found its equilibrium when the NiII atom was almost aligned with the
potassium channel. Therefore, together with the presence of π–π and elec-
trostatic interactions, the results confirmed that the presence of a metal
center improves the stabilization of the molecule onto the external face of
the G-quadruplex.
Similarly, Terenzi et al. have reported the synthesis of two
nickel(II) and copper(II) complexes of a Salen-like ligand (34 and 35 in
.
b

Figure 5.14.    (a) UV–Vis titration of a nickel(II) complex, 33, in presence of human telo-
meric G-quadruplex DNA; (b) FRET melting assays of c-KIT G-quadruplex DNA in pres-
ence of the derivative 34; (c) molecular dynamic simulation of 33 with human telomeric
G-quadruplex DNA; (d) docking simulation of 34 with c-KIT G-quadruplex DNA. (a), (c):
Adapted with permission from Ref. [71]. Copyright (2016) Elsevier; (b), (d): Adapted with
permission from Ref. [72]. Copyright (2016) Published by The Royal Society of
Chemistry.

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Targeting G-quadruplex DNA as Potential Anti-cancer Therapy 155

Figure 5.13) [72]. Differently from derivative 33, both nickel(II), 34, and
copper(II), 35, complexes are not characterized by the presence of an
aromatic ring on the N–N bridge. Spectrophotometric titrations revealed
that 34 possesses the best G-quadruplex stabilizing capabilities, espe-
cially toward the c-KIT sequence. Moreover, none of the complexes
interacts with duplex DNA. This last result clearly pointed out that both
derivatives were capable to discriminate between the double-helical and
the G-quadruplex secondary structure of the polynucleotide, therefore
.

acting as selective G-quadruplex binders. Notably, by means of molecular

modeling approaches the authors realized that the mechanism of action of
34 was different from the known G-quadruplex top/end stackers. Indeed,
the computational investigation points out the occurrence of groove
­binding of 34 into the characteristic cleft of c-KIT G-quadruplex
(Figure 5.14), thus explaining its preferential binding towards the latter
G-quadruplex rather than any other and duplex DNA. Lastly, 34 in pres-
ence of a lipophilic carrier showed a stronger cytotoxic activity than 35,
reproducing in vitro the same results achieved with G-quadruplex affinity
studies [72].

5.5  Conclusion and Perspectives

The localization in human telomeres and in oncogene promoters of
G-quadruplex DNA has attracted the attention of many researchers seek-
ing for selective anti-cancer molecules. Notably, these secondary struc-
tures have been found to be involved in other regulatory cellular processes
such as DNA replication or translation of mRNAs [2,3]. For example, they
have been found in 5′-untranslated regions (5′-UTR) of mRNAs where
b

they play a role discriminating whether the binding of the polynucleotide

with the ribosome will occur or not, thus regulating the transcription of a
protein. Interestingly, recent structural studies have allowed stating that
also Zika’s virus 3′-UTR RNA might adopt stable parallel G-quadruplex
structures, opening the way to new possible medical treatments [73].
In 2016, a large amount of G-quadruplex agents as anti-cancer scaf-
folds has been reported in the literature. Although these have not yet been
tested in vivo, they represent a valuable database of promising candidates
that might be chemically modified to improve their G-quadruplex binding

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156 R. Bonsignore, E. Trippodo and G. Barone

capabilities and enhance their biological activities. In this context, it is to

be expected that an important role in designing novel G-quadruplex bind-
ers will be increasingly played in the near future by computational meth-
ods. Overall, several steps are still needed to take before finding a
G-quadruplex binder in the shelves of a pharmacy. Nonetheless, the dis-
coveries reported so far allow being optimistic and represent a milestone
in the search of this aim.
.

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M.-P., Marinetti, A., and Bombard, S. (2016). Linking of antitumor trans
NHC-Pt(II) complexes to G-quadruplex DNA ligand for telomeric targeting.
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62. Palm, W., and de Lange, T. (2008). How shelterin protects mammalian tel-
omeres. Annu. Rev. Genet. 42, 301–334.
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B., Gonzalez-Garcia, J., Ravera, M., Vilar, R., and Paulo, A. (2016).
Anthracene-terpyridine metal complexes as new G-quadruplex DNA bind-
ers. J. Inorg. Biochem. 160, 275–286.
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64. Duskova, K., Sierra, S., Arias-Pérez, M.-S., and Gude, L. (2016). Human
telomeric G-quadruplex DNA interactions of N-phenanthroline glyco-
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b

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Chapter 6
y.

DNA-aided Super-resolution
Bioimaging

JiaJun Li*,‡, Giuseppe Arrabito† and ZhaoShuai Gao*

*Shanghai Institute of Applied Sciences

Chinese Academy of Sciences, Jiading Qu,
Shanghai Shi, China
†
Department of Physics and Chemistry
University of Palermo, Viale delle Scienze
Parco d’Orleans II, 90128 Palermo, Italy
‡
jiajunli@sinap.ac.cn

This chapter reports on the truly-exciting applications of DNA

by

­nanoscience in superresolution optical microscopies, showing the ability

provided by DNA nanostructures in solving the problems related to the
technical difficulties in implementation and ability to achieve higher
specificity and multiplexing.

6.1 Introduction
One of the key challenges in bioanalytical sciences is to identify a large
number of different molecular species with high temporal and spatial

163

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164 J. Li, G. Arrabito and Z. Gao

resolution. DNA nanotechnology is able to provide highly sophisticated

molecular tools that can facilitate imaging of biological systems with
unprecedented resolution and specificity. By employing DNA nanotech-
nology, one can design fluorescent barcodes with unprecedented spatial
control over the fluorophore feature spacing, permitting to produce multi-
plexing in barcodes, thanks to intensity-coding or geometrical coding. For
example, Lin et al. [1] reported on the fabrication of DNA-origami based
y.

fluorescent barcodes with up to dual-labelled zones to generate six pseu-

docolors (B, R, G, BG, BR and GR) from three different fluorophores,
thus generating 63 = 216 different barcodes.
Biological systems exhibit complex structures at the nanoscale.
Understanding the spatial arrangement of their individual components is
critical for unravelling the molecular mechanism that underlies complex
molecular behavior. Biological specimens can be imaged using Atomic
Force Microscopy (AFM) or electron microscopy with a typical resolution
in the nanometer and even sub-nanometer regime. However, these meth-
ods lack, so far, the ability of non-destructive monitoring of biologically
relevant dynamic processes and the determination of kinetics in the sub-
second range which can easily be accessed by fluorescence spectroscopy.
Although high-speed AFM is able to perform imaging at high speeds [2],
it is still limited to a small observation area and is in general much more
invasive compared to fluorescence microscopy methods. In principle,
while the implementation of optical microscopy approaches with high
temporal resolution is straightforward, the possibility to record data with
high spatial resolution and over long periods of time due to the diffraction
limit and photobleaching has constituted a big challenge for nanometer-
scale imaging.
by

The breakthrough in super-resolution fluorescence microscopy per-

mits today imaging and analyzing biological systems below the classical
diffraction limit by switching molecules between fluorescence on- and
off-states to obtain sub-diffraction image resolution. In fact, super-­
resolution fluorescence techniques have bypassed the traditional diffrac-
tion limit and demonstrated imaging resolution down to 10–20 nm [3–11].
By pushing optical resolutions down to the molecular scale, super-resolu-
tion optical microscopy techniques has permitted to revolutionize biomo-
lecular imaging in cells [12]. Super-resolved images obtained from single

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DNA-aided Super-resolution Bioimaging 165

molecule detections are in fact the collection of the super-localizations of

single emitters imaged successively at low density on the specimen of
interest. Here we briefly report on super-resolution fluorescence tech-
niques and the innovations brought in this research field by the application
of molecular tools derived from DNA nanotechnology.

6.2  Diffraction Limit

y.

In microscopy, resolution is one of the most important features. In com-

mon sense, it means how small an object can be “seen” by a microscope.
In a very basic light microscope, the magnified virtual images are created
by the combination of an objective lens and an eyepiece lens. By changing
the objective lens, the object can be magnified to different levels. Now
someone may ask if the object is too small to be observed why not use a
bigger magnification objective lens? In a perfect world, lens can focus a
beam of light to an infinitely small spot. In that case the intensity of the
light in that spot is going be infinitely high and it can destroy everything.
You never see any magnifier can do such thing like that don’t you? The
reason for that is the existence of the optical diffraction limit.
The optical diffraction limit was first found by Ernst Abbe in 1873
[13]. It determines the size of the focal spot (as known as the Point Spread
Function, i.e. PSF) when light with certain wavelength passes through an
optical lens. It can be described as in the equation below.

λ
d=
sinθ
by

In this equation, d is the radius of the PSF, λ is the wavelength of light

and sinθ is the numerical aperture (NA) of the objective lens. Both the
wavelength and NA have their limitations. In fluorescence microscope
the signal wavelength is the emission spectrum of the fluorescence mol-
ecules and it is usually located in the visible spectrum (450–700 nm). The
highest NA objective that can be purchased is featured with a NA of about
1.4. Then it is not hard to find the resolution limit for a conventional opti-
cal microscope is at around 200 nm. This resolution is good enough for

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166 J. Li, G. Arrabito and Z. Gao

most of the cell structure imaging but for cell organelles imaging [14] or
DNA nanostructures imaging it can hardly qualified for the jobs. There
are several techniques tried to break the law of diffraction limit, such as
near field microscopy [15], 4pi microscopy [16] and even X-ray micros-
copy [17], but none of them really succeeded. In these techniques, they
either enlarged the NA or reduced the wavelength to push their resolution
beyond the 200 nm barrier but they still work under the diffraction limit.
y.

As a matter of fact until now scientists are still not be able to truly break
this law. In the last few decades, there are three super resolution micro-
scope techniques did successfully “avoided” the diffraction limit and
pushed the resolution of the optical microscopy far beyond the 200 nm
barrier. Now all of them are commercially produced by the microscope
companies and widely used in the bio-imaging area. We are going to
introduce the three major super resolution microscopes in the sections
below.

6.3  Stimulated Emission Depleting Microscope

The first super resolution microscope we are going to introduce in this
book is the stimulated emission depleting (STED) microscope (Figure
6.1). STED microscope is the first super resolution microscope that actu-
ally pushed the resolution beyond the optical diffraction limit. Before we
talk about the principle of the STED microscope, let us first recall the
resolution of a laser-scanning microscope (LSM). The principle of the
LSM is to use a highly focused laser beam raster scan across the sample
pixel by pixel in x, y directions by using a scanning stage. The laser wave-
length is carefully chosen to fit the fluorescence excitation spectrum so it
by

can excite the fluorescent molecules to emit a red shifted fluorescent

signal. A single pixel photon sensor such as photon multiplier tube (PMT)
or avalanche photodiode (APD) is used as a detector to sense that signal
in current mode, voltage mode or photon counting mode to create a con-
trast image. It is not hard to imagine a picture filled with smaller pixels is
going to present more details of the actual sample. As described in the
previous section, the size of the PSF is limited by the optical diffraction
limit, which is about 200 nm, meaning that any object smaller than
200 nm is going to looks like a 200 nm Gaussian spot in the optical

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DNA-aided Super-resolution Bioimaging 167

(a) (b)
y.

(c) (d)

(g) 1.1 Confocal

(e) (f) 1.0
STED
Fit STED
Fit Confocal
0.9
Normalized Fluorescence

0.8

0.7

0.6

0.5
by

0.4

0.3

0.2
0 100 200 300 400 500 600 700
Relative Displacement

Figure 6.1.    The images above are made from a partially customized STED microscope.
The donut shape STED depletion beam PSF (red) overlapped on the excitation beam PSF
(green) in lateral (a) and longitudinal, (b) directions which enhance the resolution of a
confocal image, (c) beyond the diffraction limit (d). (e) and (f) are the zoom of the white
squares in (c) and (d). (g) is the intensity profile of a single fibril (alone green lines in
(e) and (f)).

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168 J. Li, G. Arrabito and Z. Gao

microscope. Is there any chance to surpass this limit and confine the PSF
beyond the 200 nm? The answer is yes and no. In 1994, Prof. Stefan Hell
from the Max Planck Institute for Biophysical Chemistry in Göttingen,
Germany brought up an idea to use a depletion beam to compress the
efficient excitation PSF to a sub-diffraction limit size. In 1999, he made
the first STED microscope and successfully increased the resolution of
the optical microscope to 105 nm [18], which was the first time optical
y.

microscope resolution went beyond the diffraction limit barrier. Because

of this achievement, Prof. Stefan Hell was awarded a Nobel Prize in
Chemistry in 2014.
The secret behind the STED microscope is an optical phenomenon
called stimulated emission. It happens when a depletion laser PSF spa-
tially and temporally overlapped on an excitation beam PSF. The wave-
length of the depletion laser was carefully chosen to be at the end of the
fluorescence molecule’s emission spectrum. The stimulated emission can
compete with the fluorescence emission. If the depletion beam intensity is
high enough it is possible to totally eliminate the fluorescence emission.
If this happens we say the fluorescence molecule is “switched off” by the
depletion beam. Besides of the intensity and the wavelength another
important property of the depletion beam is its shape. A classic Gaussian
shaped depletion beam can only increase the resolution of the image in
one dimension by overlap it on the side of the excitation beam PSF. In the
very early stage, Prof. Hell suggested using four depletion beams to
squeeze the efficient excitation PSF in a very small point, this plan dra-
matically increased the complexity of the microscope and it was never
been realized. Until later, vortex phase plate was invented, this phase plate
can introduce a 0–2π phase delay into the laser wavefront and eventually
by

cause the PSF at the focal point become a ring shape or donuts shape
which has a zero-intensity region in the center. Now once this PSF over-
lapped on the excitation PSF, it “switches off” all the fluorescence mole-
cules except the ones in the zero-intensity center [19]. By overlapping this
donut shape depletion beam onto a confocal microscope excitation laser,
it can upgrade the confocal microscope to a STED microscope.
Therefore the resolution of the STED microscope is now no longer the
size of the excitation PSF, it is depending on the size of the depletion

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DNA-aided Super-resolution Bioimaging 169

beam’s zero-intensity center [20]. The new resolution equation can be

written as

λ
d=
I
n sinα 1+
I sat
y.

In this equation, λ is the wavelength of the fluorescence signal, n sinα is

the NA of the objective lens, I is the intensity of the depletion beam and
Isat is the saturation intensity of the florescence molecule. From the equa-
tion we can see that it is very similar to the classic diffraction limit equa-
tion except the terms of I and Isat. In theory, if the depletion laser intensity
can reach infinitely high, the resolution of the STED microscope is unlim-
ited. In practice it is impossible, not only because there is no infinitely
high intensity laser, but also when the laser intensity reaches certain level,
it can cause some serious damages to the sample [21, 22]. Therefore it is
hard to define an exact number for STED microscope resolution. Usually
commercial STED microscope claims their system can reach a resolution
of 40 nm, which will depends on what kind of sample you are imaging.
The other term in the STED resolution equation is the saturation intensity
Isat. It is an instinct property of fluorescence molecules, which is very hard
to change, unless to leave the fluorescence molecule in an extremely
low temperature. A recent research has been studies that in liquid
helium (about -269°C) the resolution of the STED microscope can reach
sub 10 nm [23].
Depending on the laser source used for depletion, the STED micro-
scope can be further classified into pulsed laser STED microscope or
by

continue wave laser STED (CW-STED) microscope. The pulsed laser

STED microscope is the original design of the STED microscope. It has
the benefits of high resolution and low sample damage. In 2007, the
CW-STED microscope was invented [24]. The propose of this design is to
simplify the complexity of the STED microscope setup by avoiding pulse
temporal synchronization. Since CW-STED requires a much higher aver-
age intensity depletion laser, this design didn’t get widely used. In 2008,
a supercontinuum laser was used for excitation source and depletion
source, the design gave STED microscope the capability to easily pick the

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170 J. Li, G. Arrabito and Z. Gao

suitable excitation/depletion wavelength pair [25]. Time gating STED

(gSTED) microscopes is another member of the STED microscope fam-
ily. It was first time invented in 2011 [26]. In gSTED microscope, a pulsed
laser is used as excitation source and a CW laser is used as depletion
source, so there is no need for temporal synchronization. A detector with
time gating function was used to remove the fluorescent signal from the
first few nanoseconds after the excitation pulses arrive. These signal car-
y.

ries non-superresoltuion information which can reduce the image quality.

Due to this design, the STED setup was simplified and its resolution
become comparable to the pulsed laser STED microscope. This design
now is used in Leica commercial STED microscopes.

6.4  Localization Microscopes

Localization microscopes are another group of super resolution micro-
scopes widely used in bio-imaging area, which include stochastic optical
reconstruction microscope (STORM) and photoactivated localization
microscope (PALM). They both share a very similar principle. From hard-
ware point of view, the two microscopes are totally identical, the only
difference lies in the fact that the PALM was described using photoswitch-
able fluorescent proteins [6], whereas the STORM was originally described
suing Cy5 and Cy3 dyes attached to nucleic acid or proteins. In principle
any photoswitchable fluorophore can be used, and the STORM has been
demonstrated with a variety of different probes and labeling strategies. The
STORM microscope was invented by Prof. Xiaowei Zhuang from Harvard
University in United States [27]. The principle of this super resolution
technique is totally different to the STED microscope we described in the
by

previous section. If on one hand, the STED microscope is a super resolu-

tion microscope modified from a confocal laser scanning microscope by
modifying its hardware, the STORM microscope is a super resolution
microscope modified from a total internal reflection fluorescence
(TIRF) microscope by adding some specific algorithms in it (Figure 6.2).
TIRF microscope is a very commonly used fluorescence microscope. The
difference between TIRF and a wide field fluorescence microscope is the
way it illuminates the sample. A wide field fluorescence microscope illu-
minates a sample by sending the excitation beam directly onto it, by doing

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DNA-aided Super-resolution Bioimaging 171

Fluorophores too Stochastic activation and Super-resolution image

dose to resolve localization of individual molecules reconstructed from localizations
y.

TIRF STORM

Figure 6.2.    The principle of the STORM microscope is to calculate the localization of
every fluorescent molecules (upper). After the image reconstruction, the resolution is dra-
matically enhanced compare the to the conventional TIRF microscope (lower).

this the fluorescent molecules emit the florescent signal and these signals
are received by a CCD or CMOS camera. A disadvantage of the wide field
fluorescence microscope is during the imaging process the entire sample
is illuminated by the excitation source. The fluorescent molecules from
different layers of the sample produce huge amount of signal and the
detector receives these signals spontaneously. The microscope couldn’t
by

distinguish which layer does the signal come from. This significantly
decreases the signal to noise ratio, especially in the thick tissue imaging.
In TIRF microscope the illumination source is the evanescent wave of total
internal reflection (TIR). The TIR happens when the laser incident angle
went beyond the critical angle (Snall’s law). This evanescent wave is only
few hundreds thick and only illuminates a very thin layer of sample. This
technique reduces the image background noise and enables single mole-
cule fluorescence imaging. The capability of the single fluorescence mol-
ecule imaging is the key to the STORM super resolution microscopy.

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172 J. Li, G. Arrabito and Z. Gao

As described in previous sections, no matter how small the objects

are, if the sizes of them are smaller than 200 nm, in an optical system they
only can be seen as a 200 nm Gaussian spot. Though the size of the PSF
cannot be changed, it is still possible to localize its center position. The
standard error of the localization can be described in the equation
below [28].
y.

 s 2   a 2 / 12   8π s 4 b 2 
σ =   +  + 
 N   N   a2 N 2 

In this equation, s is the standard deviation of the PSF Gaussian function,

N is the number of photons captured from the single fluorescent molecule,
a is the pixel size of the detector and b is the standard deviation of the
background. Once the photon number reaches 1000, the equation can be
approximated as

σ ≈ s/ N

Therefore now the standard error is only related to the standard deviation
of the PSF Gaussian function and the number of photons captured from
the single fluorescent molecule. In an ideal world, if a fluorescent mole-
cule stays absolutely immobilized and can produce infinite number of
photons, it can be localized to an infinitely small spot, sometimes even
smaller than the size of the single molecule itself. Even in practice it is
still possible to keep the localization distribution in a few nanometer
scale, which is much smaller than the diffraction limit. This method is
ideal for single fluorescent molecule tracking but still not good enough
by

for super resolution imaging. In fluorescence imaging the fluorescent

molecules are distributed very close. If the distance between two fluores-
cent molecules is smaller 200 nm and they are excited spontaneously,
they cannot be resolved in a single frame, which makes single molecule
localization impossible. Therefore another key component in the STORM
microscope is to make fluorescent molecules “blink,” so they can be
resolved temporally. The situation can be described as in a paintball game
if one shoots all the bullets close to the center of a bullseye, eventually he

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DNA-aided Super-resolution Bioimaging 173

will get a paper full with paint spots. Since those paint spots are so close
and overlapped on each other you are not able tell the localizations of
each bullet. If at the meantime, one used a high speed camera captured
the whole shooting process, it is possible to stop at each frame when a
single bullet splashed on the paper and calculate the localization of its
“PSF.” By analyzing every frame it is possible to draw all the bullets
localizations on a new paper. This is the secret behind the STORM
y.

microscope.
The trick is to control the fluorescent molecules “blink” rate to make
sure there is a very low possibility two close fluorescent molecules be
excited spontaneously in a single frame. There are a lot of commercial
fluorescent dyes on the market which are designed for this purpose [29],
which covers almost all the common spectrums.
In order to obtain a decent super resolution image usually 30,000–
50,000 frames are needed. Just like STED microscope, its super resolu-
tion efficiency is highly dependent on the sample and the fluorescent
dye. In most of the cases the resolution can reach up to 20 nm [30, 31],
which is slightly better than the STED microscope. One has to consider
that the imaging time is extremely long, which makes the STORM
impossible to do in vivo imaging. Since the STORM microscope’s illu-
mination technique is based on TIRF illumination, it was considered
lack of the 3D imaging capability. In recent years, some microscope
company use a cylindrical lens with 3D algorithm realized the STORM
whole cell imaging.

6.5  Structured Illumination Microscope

by

The last super resolution microscopy that we here describe is the SIM
microscope. The Structured Illumination Microscope (SIM) microscope
was invented by Prof. M. G. L. Gustafsson in 2000 [29]. The principle of
SIM microscope is the combination of structured illumination and Fourier
transform [32]. With respect to most of the microscopes, the SIM’s illu-
mination source is a laser with a sinusoidally striped illumination patterns.
When this pattern overlaps on an unknown pattern e.g. the structure of the
fluorescent molecules distribution from the sample, it produces moiré

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174 J. Li, G. Arrabito and Z. Gao

y.

Figure 6.3.    When two structured illumination overlap on each other in certain angle (one
for the sample (a) another one for the structured illumination source (b)), they result a
moiré fringes (c). An extremely fine sinusoidally pattern (200 nm between each line) cre-
ates two bright spots and an origin in its Fourier transform image. The distance between
the two bright spots determines the size of the observation region (a). The moiré fringes
provides information outside of the observation region (b). By rotating the sinusoidal
illumination several time radius of the observation region of the optical microscope can be
by

doubled therefore it increases the microscope resolution.

fringes. Moiré fringes are enlarged scale interference patterns, which

usually happen when two unidentical patterns overlap on each other as
shown in Figure 6.3(a). This enlarged pattern contains information of both
the illumination pattern and the sample pattern.
Under the diffraction limit, if sample structure is smaller than 200 nm,
it cannot be resolved by the optical microscope, but when they multiplied

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DNA-aided Super-resolution Bioimaging 175

with the structured illumination source the moiré fringes may become
observable. Since one already knows the structure the illumination source
and the moiré fringes, we can extract the structure of the sample. Different
to the previous two super resolution microscopes, the resolution of the
SIM microscope is settled. In image processing, all images can be trans-
formed to its Fourier transform, a mathematical function that transforms
image from its spatial domain to its frequency domain. In frequency
y.

domain the high frequency components (the components away from the
origin) determines image’s “detail” and the low frequency components
(the components close to the origin) determines image’s “gray scale.” If
one has a sinusoidally pattern as image, in its Fourier transform image it
is presented as two mirrored bright spots offset away from the origin. The
directions of the two spots are respect to the directions of the strips of the
pattern and the distance between the spots to the origin are proportional to
the inverse line spacing of the pattern. If the lines spacing are exactly at
the diffraction limit and rotating extremely fast, in its Fourier transform
image it is presented as a circle constructed by the two bright spots. The
components within this circle are called the “observation region” within
the optical diffraction limit. Since the higher frequency components deter-
mine image’s detail, all the components outside of this circle are “super
resolution region.” In SIM microscope the moiré fringes provide informa-
tion outside of the observation region. In its Fourier transform, two extra
bright spots are centered by the two original bright spots. Since the two
original bright spots are already at the edge of the observation region, by
the rotating the direction of the sinusoidally striped illumination several
times (usually 3 times) the SIM microscope is able to double the resolu-
tion of the traditional optical microscope in x, y directions.
by

6.6 Structured Overview about Super

Resolution Technologies
The three super resolution technologies described in the previous sections
are the most widely used super resolution microscopes. All of them are
commercially produced and have been served in the bio imaging area for
the last a few years. There is no simple way to say which microscope is the
best. All of them have their own pros and cons. It is important to choose

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176 J. Li, G. Arrabito and Z. Gao

the right microscope, which ideally fits samples condition [33]. Here is a
table may give users some ideas to the most suitable microscope.

STED STORM SIM

Resolution 60 nm 40 nm 100 nm
Speed Fast Slow Very fast
In vivo imaging Possible Impossible Possible
y.

Damage to the sample High Low Low

3D imaging Possible Possible Impossible
3D Super resolution Possible Possible Impossible
Multi-color imaging Conditions may applied Possible Possible

6.7 DNA-nanostructures Aided Super

Resolution Imaging
There are many parameters which affect the performance of current super-
resolution techniques in imaging complex biological systems. Just to
mention the most important factors, one can consider the fluorophore
photon budget, suboptimal fluorophore imaging efficiency and the limited
control over target blinking kinetics. Such restrictions bring to limited
photon count per localization, limited number of blinking events per target
and high fraction of false localizations which finally lead to limited num-
ber of visualized blinking events per target, affecting signal-to-noise ratio
and the visualization of individual targets within dense clusters. Methods
such as PALM or STORM have difficulties to access very densely labeled
regions due to spontaneous photoswitching, which prevents imaging indi-
by

vidual fluorophores in these regions [34].

In the following, we describe two super resolution microscopy tech-
niques in which DNA nanostructures play a fundamental role. In particu-
lar, we will describe Binding-activated localization microscopy (BALM)
and Points accumulation for imaging in nanoscale topography (PAINT).

6.7.1  Binding-activated localization microscopy

BALM [35], is a superresolution technique in which free solution diffusing
dyes turn bright upon binding to a target structure and can be localized with

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DNA-aided Super-resolution Bioimaging 177

y.

Figure 6.4.    Scheme of BALM. Dyes diffusing in solution phase turn bright when they
bind to DNA target structures. Binding kinetics is determined by the on rate kon, whereas
detaching is determined by koff. Reprinted with permission from Ref. [35]. Copyright
(2011) American Chemical Society.

sub-diffraction resolution before they eventually bleach or detach. Upon

binding to target object, the chromophores become immobilized and the
quantum yield increases of 2–3 orders of magnitude (Figure 6.4).
In their paper [35] Schoen et al. improved upon both localization
accuracy and density by utilizing fluorophores that are “switched on”
upon binding to a target structure and directly exploit this property to
localize them under dynamic binding conditions (Figure 6.5). By iterating
cycles of binding, localization and bleaching, it is possible to reconstruct
the biological structure under investigation. Such strategy is defined as
BALM and can be generalized to visualize biological or synthetic struc-
tures with nanoscale resolution using a variety of available dyes. By the
employment of a dynamic labeling scheme and the optimization of fluo-
by

rophore brightness, they obtained a resolution of ~14 nm (fwhm) and a

spatial sampling of 1/nm.
The authors used YOYO-1 dye to image double-strand DNA with
unprecedented high localization accuracy and high labeling density at the
same time. Subsequently, they used the dye PicoGreen to image chromo-
somal organization in fixed Escherichia coli cells, obtaining a dramatic
improvement in resolution when compared with diffraction-limited
microscopy. The chromosome in such cells and the overall contour of the
nucleoid inside the cells obtaining details with a resolution on the order of
about 100 nm.

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178 J. Li, G. Arrabito and Z. Gao

y.

Figure 6.5.    Imaging of chromosomes in fixed bacteria by BALM with PicoGreen fluo-
rophore. (a) Image obtained via BALM, (b) diffraction-limited image, (c) image of bacte-
ria during division, (d) enlarged view. Scale bars: 1 mm (a)–(c), 200 nm (d). Adapted with
permission from Ref. [35]. Copyright (2011) American Chemical Society.

6.7.2  Binding-DNA-aided point accumulation for

imaging in nanoscale topography
6.7.2.1  Points accumulation for imaging in
nanoscale topography
by

In the PAINT method [11], the object to be imaged has to be continuously

targeted by fluorescent probes which are present in the solution phase.
Notably, fluorescent probes flux on the object is dependent on molecular
diffusion coefficient and the concentration gradient of the probes. A fluo-
rescent signal on the object to be imaged is constituted by a diffraction-
limited spot. Such signal can be destroyed if the fluorescent probe is
photobleached or if it dissociates from the object. In this original version
of PAINT, the probe does not necessarily have a specific interaction with

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DNA-aided Super-resolution Bioimaging 179

the object, this meaning that such interaction can be electrostatic cou-
pling, hydrophobic interaction. The authors also showed that the colli-
sion rate of probes with the object depends linearly on the concentration
of the probes. Such fluorescence spikes appearance can be approximated
by the diffusion-controlled bimolecular reaction rate constant multiplied
to the probability that a collision is able to generate a binding configura-
tion leading to a fluorescent signal. As one can expect, the nature of local
y.

discrimination in imaging is dependent on the specific interaction

between fluorescence probe and the object.
In PAINT, it is necessary to keep density of probes on the object sur-
face below the value of one molecule per squared micron in each observa-
tion period. For this reason, the obtained fluorescent image is treated as a
PSF from a single molecule. In order to achieve this condition, the time
between subsequent observation frames has to be longer than the time of
fluorescence burst of probe and by maintaining the image frame length
shorter than the period between collisions.
Probing molecules are deactivated between successive image record-
ings. The authors demonstrated the impressive 25 nm resolution on lipid
bilayers, contours of such bilayers and contours of such lipid bilayers and
large unilamellar vesicles. In particular, they used a fluorescently labeled
transferrin protein and a small environment-sensitive dye (Nile red) to
demonstrate the possibility to use different probes by employing this
method. Remarkably, PAINT can be used without any modification of the
imaging objects such as cellular compartments or organelles or cellular
membranes. By varying the probe molecule, it is possible to obtain selec-
tivity in imaging specific objects. For instance, many fluorescently labeled
proteins which have high specificity to certain cellular compartments can
by

be used as imaging probes.

In other words, the number of molecules targeting a circular area πR2
in unit time from a solution which contains N molecules per unit volume
is given by a Smoluchowski type relation, 4DRN (s-1), in which D stands
for molecular diffusion coefficient. Therefore, in the steady state, the
number of probes on the object is given by 4DRN <τon>, in which <τon>
is the mean time that the probe spends with the object before it dissociates
from it or it is photobleached. The deactivation rate <τon>-1 is defined as
the sum of the mean rates of photobleaching and dissociation of the probe

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180 J. Li, G. Arrabito and Z. Gao

from the object, by assuming that the probes act independently of one
another. Bleaching rate is a function of the excitation intensity and the
photochemical and photophysical properties of the probe in the surround-
ing medium. As expected, it is possible to control the time that the probe
spends in contact with the object by varying the concentration of the probe
in solution. Since the number of probe molecules in the surrounding solu-
tion is not significantly depleted by the photobleaching occurring on only
y.

one small illuminated object, it is possible to acquire the image by accu-

mulating as many images frames as needed. The image is formed by
overlapping such frames. The total recording time is limited by the
mechanical stability and movements of the object to image with respect to
the light collection system.

6.7.2.2  DNA-aided PAINT

As previously explained in PAINT approach, imaging is carried out by
the employment of diffusing fluorescent molecules that interact tran-
siently with the sample. A key limit of PAINT is that interaction of dyes
with the sample occurs via electrostatic or hydrophobic coupling, so it
cannot be used in multiplexed analysis of different biomolecules in the
same sample. In order to achieve programmable multiplexed dye inter-
actions and to increase the specificity and the number of usable fluoro-
phores, DNA-PAINT was developed [36, 37]. In DNA-PAINT, stochastic
switching between fluorescence on- and off-states is executed via tran-
sient binding of fluorescently labeled oligonucleotides (“imager”
strands) to complementary “docking” strands on DNA nanostructures. In
the unbound state, it is possible to observe only background fluores-
by

cence from imager strands. In case of binding and immobilization of an

imager strand, fluorescence emission is detected. In DNA-PAINT, the
transient binding of fluorophore-labelled imager strands to target-
bound docking strand permits to achieve the necessary blinking condi-
tion for superresolution reconstruction. Remarkably, the continuous
replenishment of imager strands renders DNA-PAINT immune to pho-
tobleaching, a­ llowing high localization precision by extracting a large
number of photons per single-molecule localization and a high target

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DNA-aided Super-resolution Bioimaging 181

y.

Figure 6.6.    Scheme of DNA-origami structures (nominally 291.5 × 23 nm) employed for
the single-molecule binding. Each structure contains a fluorescently labeled staple strand
(labeled with ATTO532). The docking strand extension is shown at the center of the structure.
Without binding of the imager strand, no fluorescence signal is observed. (a) Without binding
of the imager strand, no fluorescence signal is observed. (b) Upon hybridization of an imager
strand, fluorescence signal was observed in the red channel. (c) Trace of fluorescence inten-
sity vs. time for the unbound state (τd is the time for the dissociated state). Adapted with
permission from Ref. [36]. Copyright (2010) American Chemical Society.

separability by collecting a large number of blinking events from each

target. Additionally, due to independent and programmable control of
blinking on- and off- rates, DNA-PAINT permits low imaging back-
ground in dense clusters from appropriately adjusted blinking duty cycle
by

based on the target density.

In a fundamental paper, Jungmann et al. [36] implemented the PAINT
concept by means of reversible labeling with diffusing DNA probes (see
Figure 6.6). They defined this approach as DNA-PAINT. This uses fluo-
rescently labeled single stranded DNA probes in solution that hybridize to
complementary single-stranded extension on DNA-origami structures,
thus imaging them in real time. Single-molecule DNA binding and
unbinding events are directly visualized using fluorescence microscopy,

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182 J. Li, G. Arrabito and Z. Gao

making DNA-PAINT, apart from its imaging capabilities, an ideal tool for
analyzing, e.g. hybridization kinetics on DNA structures.
They fabricated long rectangular 2D origami structures with dimen-
sions of about 290 × 23 nm. Each origami structure is decorated with a
fluorescently labeled staple strand (ATTO532 labeled) and one or more
docking strands. They firstly acquired a diffraction-limited fluorescence
image using the directly incorporated fluorescent label (here ATTO532
y.

using an excitation wavelength of 532 nm) to determine the positions of

the origami structures on the surface. Then, they added ATTO655 labeled
imager strands having a complementary sequence to the docking strands.
They selected lengths of the imager strands just to result only in short-
term binding events at ambient conditions. A second excitation wave-
length (650 nm for ATTO655) is used to monitor the transient binding of
single imager strands to the DNA origamis. From the measurements, they
could assign low signals corresponding to unoccupied staple strands,
whereas binding of an imager strand resulted in fluorescence increase.
The perfect superposition of the green marker image (ATTO532) with a
standard deviation image that highlights the imager strand (ATTO655)
locations based on the maximum signal variations indicates the high
selectivity of binding to DNA origami. Given the possibility to monitor
individual hybridization events on a DNA origami in real time, they could
calculate the association rate from the τd, which is defined as the mean
interevent lifetime (i.e. the dissociated time) since 1/τd = konc, in which c
is the imager strand concentration.
They could also determine imaging efficiency, given the precise con-
trol over number and position of docking strands on monomeric DNA-
origami structures. The authors could determine the fraction of docking
by

strand positions that could be imaged. The imaging efficiency is actually

an open challenge for superresolution approaches, for many reasons like
not precise labeling in some positions, bleaching of fluorophores during
photoactivation or not sufficient number of emitted photons before pho-
tobleaching. For example, in PALM, only a percentage of about 30% of
molecules is usually analyzed [6]. The authors could determine that
almost all docking strands can be imaged by comparing the presence of
staple strands at specific positions using AFM and DNA-Paint measure-
ments. In this regard, after having introduced three biotinylated DNA

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DNA-aided Super-resolution Bioimaging 183

strands in the DNA origami, streptavidin was added to the origami sample
on a mica surface and incubated for several hours. They imaged structures
containing three or two streptavidin molecules, since only these structures
can be counted by DNA-PAINT. Remarkably, the efficiency of imaging
was found to be almost complete (around 95%), this value is significantly
higher than current super-resolution techniques.
Jungmann et al. reported on an unprecedented methodology that car-
y.

ries out the transient binding of short fluorescently labeled oligonucleo-

tides (DNA-PAINT, a variation of point accumulation for imaging in
nanoscale topography) allowing for multiplexed super-resolution imaging
at sub-10 nm spatial resolution in vitro on synthetic DNA structures [38].
They demonstrated superresolution imaging of microtubules inside HeLa
cells using Atto 655-labeled imager strands. Remarkably, they defined a
multiplexing approach (Exchange-PAINT) since it allows sequential
imaging of multiple targets inside single cells using only a single dye and
a single laser source. In exchange-PAINT only one fluorophore is
employed, thereby allowing the selection of an optimal dye with respect
to its photophysical properties for superresolution imaging. They obtained
super-resolution images of β-tubulin in microtubules, COX IV in mito-
chondria, TGN46 in the Golgi complex and PMP70 in peroxisomes using
Cy3b dye (see Figure 6.7(a)).
In a very recent article, Dai et al. [39] reported on the combination of
“discrete molecular imaging” with Exchange-PAINT showing highly
accurate (<1 nm) three-color registration, in addition to highly accurate
drift correction (<1 nm r.m.s.) within each channel. The necessary condi-
tion for “discrete molecular imaging” is the high localization precision
that allows a full width at half maximum (FWHM) resolution equal to or
by

smaller than the spacing between them.

They could visualize individual targets in a compactly labeled molec-
ular grid of targets (with a point-to-point spacing of ∼5 nm) demonstrating
multiplexed imaging on a three-color nanodisplay board with ∼5 nm
­pixels. They showed the potentiality of their method by imaging a custom-
designed letter pattern (“Wyss!”) on a 60 × 85 nm origami nanodisplay
breadboard with 5 nm display pixel size. Finally, they imaged a three-
color mixture structure of the 5 nm grid with multiplexed DMI with an
average localization precision of 2.0 nm (see Figure 6.7(b)). Their

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184 J. Li, G. Arrabito and Z. Gao

y.

Figure 6.7.    (a) Multiplexed cell analysis by DNA-PAINT. Each target in a fixed HeLa is
labeled with an antibody carrying a unique DNA-PAINT docking sequence, recognized by
a specific Atto 655 labeled imager strand. Super-resolution images of β-tubulin in micro-
tubules (i), COX IV in mitochondria (ii), TGN46 in the Golgi complex (iii) and PMP70 in
peroxisomes (iv) were obtained sequentially; (v) overlay of all four targets. Scale bar is
500 nm. Reprinted with permission from Macmillan Publishers Ltd.: Nature Methods
(Ref. [38]), copyright © (2014). (b) Discrete molecular imaging with complex patterns and
multiplexed (three color) images. DNA-PAINT super-resolution images (top row) and
automatically fitted images (bottom row) are shown for all three single-color channels
(left three columns) and the combined image (right column) for two representative 5 nm
by

grid structures. Reprinted with permission from Macmillan Publishers Ltd.: Nature
Nanotechnology (Ref. [39]), copyright © (2016).

approach can potentially enable the determination of the position and

identity of discrete molecular components in complex biological or syn-
thetic nanostructured systems, so allowing for a complementary approach
to electron microscopy and crystallography with single-molecule sensitiv-
ity. According to the authors, two big issues remain for future develop-
ment of their methodology. The first one deals with the necessary trade-off
between spatial and temporal resolution, i.e. the compromise between

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DNA-aided Super-resolution Bioimaging 185

blinking times (higher times lead to higher spatial resolution) and image
acquisition time (higher times lead to slower acquisitions). The other issue
is the limited absolute labelling efficiency (i.e. average number of probes
labeled per molecular target), which can be overcome by employing
genetically labeled tags (such as SNAP-tags), aptamers, small-molecules
labels just to cite the most important ones. The future development of their
methodology allows for investigation of molecular features in diverse
y.

biological systems such as the molecular composition and architecture of

complex cellular systems (such as molecular states of individual protein
components or architecture of chromosomes) with unprecedented spatial
resolution.

6.8 Conclusion
In Conclusion, DNA nanostructures allow for a significant simplification
in currently established superresolution optical microscopies, solving the
problems related to the technical difficulties in implementation. Even
more importantly, DNA allows for truly multiplexing for a large number
of distinct biomolecular targets. From the most recent literature reports,
one can expect that in the future, it will be possible to quantitatively study
molecular features in diverse crowded environments (cytosol, cellular
membranes) allowing for unprecedented analysis of biological systems at
nanometer scale resolution.

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Index

A caveolae-mediated endocytosis, 112,

Abbe, Ernst, 165 115–116
acceptor, 65–66, 80, 82 cell lysates, 117
Archimedean tilings, 31, 38, 41 cell-SELEX, 100–101, 104
atomic force microscopy (AFM), 12, cellular environment, 116
16, 60–61, 164 circular dicroism spectroscopy, 138,
147, 152–153
B c-KIT, 132, 134, 137, 139–141, 143,
Bang, Ivar, 130 154–155
B-DNA, 6, 79 c-KIT1, 138
binders, 130, 135–138, 141, 144, 146, clathrin/caveolin-independent
148, 150–151, 155–156 endocytosis, 112
binding-activated localization clathrin-mediated endocytosis
microscopy, 176–178 (CME), 112, 114–116
biosensors, 50, 58–59, 66, 91, c-MYC, 134, 138–142, 144–149,
93–95, 190 151
BRACO-19, 136, 148 continue wave laser STED, 169
Crick, Francis, 2
C crossovers, 36, 38–39, 42–44, 46
caDNAno, 45 cubes, 118
cages, 118 cytosine-based guanine
CANADA package, 37 oligodeoxynucleotide, 103
CanDo, 45 cytosine–phosphate–guanine (CpG),
capsid proteins, 121 103, 119–120

193

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 9”x6”
b2875  DNA Nanotechnology for Bioanalysis: From Hybrid DNA Nanostructures to Functional Devices

194 Index

D E
depletion beam, 167–169 electron beam lithography, 63
dip-pen, 108 encapsulate spherical DNA-origami,
dip-pen nanolithography, 60–62, 121
106–107, 109 endocytosis, 112
DPN, 107 enzyme-linked immunosorbent assay,
DNA-aided points accumulation for 75
imaging in nanoscale topography, evanescent wave, 171
180–184 exchange points accumulation for
DNA aptamers, 12–13, 49–51, 70, imaging in nanoscale topography,
74, 100–104, 124 183
DNA conjugation, 3, 14 excitation beam, 168
DNA-directed immobilization (DDI),
105–107, 109–110 F
DNA groove, 136–138, 148–149 Feynman, Richard, 1, 4
DNA junction, 33–34 fluorescent intercalator displacement
DNA machine, 16–18 (FID) assay, 150, 152
DNA nanocarriers, 100 Förster resonance energy transfer,
DNA nanotechnology, 100 64–66, 75, 81, 83
DNA origami, vii, 3, 18, 20, 22–26, Fourier transform, 173
29–31, 42–46, 52–55, 67–71, 88, functionalization, 4, 8, 11, 14
110, 117–119, 120, 125–127, 164,
181–182, 187, 191 G
DNA–protein immobilization, 100 GIDEON, 38
DNA repair activities, 84 Gold and Szostak, 49
DNA sensors, 57, 59, 84, 93 G-quadruplex, 6–7, 130–131, 133,
DNA tiles, vii, 8, 20, 28, 53 138–147, 153
DNA walkers, 17–18 grooves, 152, 155
donor, 65–66, 80, 82 G-tetrad, 132, 147
donor–acceptor, 65–66, 80 Gustafsson, M. G. L., 173
double helix, 4–6, 8, 28, 70, 82, 130
Douglas, Shawn, 120 H
doxorubicin, 118–119 hairpin loops, 6
drug delivery, 100 hairpin motif, 6–7
drug delivery cargo, 116 hairpin structures, 6
duplex DNA, 134, 141, 144, 146, Hell, Stefan, 168
151–152, 154–155 high frequency components, 175
dynamic DNA nanotechnology, 6, Holliday junction, 8–9, 12, 24, 33,
16–17 68

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Hoogsteen base pairing, 7 N

Hoogsteen hydrogen bonds, NanoEngineer-1, 38
131–132 nanopore, 69–70
hybridization, 6 nanotechnology, vii, 1, 19, 122, 188,
191
I nanotubes, 118
i-motifs, 6–8 nucleobases, 5
in vivo imaging, 120 numerical aperture, 165–166, 169
isoquinoline alkaloids, 137
O
K observation region, 175
Klug, Aaron, 133 oligonucleotides, 14–16
optical diffraction limit, 165–166
L
laser-scanning microscope (LSM), P
166 π-stacking, 138, 140, 142, 146–147,
Liedl, Tim, 45 152
logic-gate DNA-origami nanorobot, padlock probes, 71–72, 74
120 PAINT, 188
low frequency components, 175 parts, 64
PDC, 151
M pen array, 109
macro-pinocytosis, 112 2D polymer-pen lithography
MDA–MB231, 148 (2D-PPL), 110
MDA–MB231 cells, 149 dip-pen arrays, 109
melting temperature, 79–81 polymer pen array, 110
microarrays, 104–107, 109–110 phagocytosis, 112–114
arrays, 106–110 photoactivated localization
bioarrays, 106 microscope (PALM), 170, 176,
micrometer-scale arrays, 109 182
oligonucleotide arrays, 111 pinocytosis, 112
sub-cellular scale EGF arrays, plasmonics, 66–68
110 platonic tilings, 41, 48
microcontact printing, 63–64 points accumulation for imaging in
Mirkin, Chad, 4 nanoscale topography, 176, 178–181
moiré fringes, 174 point spread function, 165, 167–168,
molecular dynamics, 136, 139–140, 172–173, 179
153–154 polymerase chain reaction (PCR),
72–73, 76–78

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196 Index

Prof. Tan, 101 smart therapeutic nanodevices, 100

protein-of-interest (POI), 14–16 stimulated emission depleting
pulsed laser STED, 169 (STED), 166–170, 173, 176
stochastic optical reconstruction
Q microscope (STORM), 170–173,
quadruplex, vii, 79, 129, 133–137, 176
140, 147, 150–152, 154–162, strand-displacement, 19
189 structured illumination microscope
quantum dots (QDs), 65–66, 75 (SIM) microscope, 173
quarfloxin, 136 super resolution microscope, 166
surface enhanced Raman scattering,
R 64, 68–69
rolling circle amplification (RCA), surface plasmon resonance, 64, 66,
72–78, 83 87
rolling circle enhanced enzyme systematic evolution of ligands by
activity detection (REEAD), exponential enrichment (SELEX),
77–78 49, 73
Rothemund, Paul, 3, 42 Szostak, Jack William, 49

S T
Salen, i.e. 2,2′-Ethylenebis(nitrilomet telomerase, 135–136, 148
hylidene)diphenol, 153–154 telomeres, 133–135, 151, 155
Salphen, i.e. N,N’-phenylenebis(salic tensegrity, 44–45
ylideneimine), 153 tetrahedron, 118–119
Seeman, Ned, 2, 9, 16, 28 tetra-(N-methyl-4-pyridyl)porphyrin,
self-assembly, 4, 10–11, 20, 24–26, 148
29, 33, 37, 41, 52–54, 88, 95, 111, thiazole orange, 150, 152
124, 190 tile assembly, 32, 36, 38, 40–41
sensor, 59, 66, 72, 75–76, 78–79, 81, tile-to-tile assembly, 27
85, 94, 102, 166 tilings, 39, 41
SEQUIN program, 33–37 topoisomerases, 75, 77–78, 84
serum, 117 total internal reflection fluorescence
Shih, William, 31, 44 (TIRF) microscope, 170
single cell, 100, 104, 106, 109–110, triple helix, 6–8
121 tumors, 120
single-strand displacement, 17–18
sinusoidally pattern, 175 U
siRNA, 103, 119–121 uniquimer 3D, 38

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V Watson, James, 2
vHelix, 48 wireframe structure, 46
virus capsid proteins, 121
vortex phase plate, 168 Z
zebrafish, 149–150
W zebrafish embryos, 149
Watson and Crick, 5 Zhuang, Xiaowei, 170
Watson–Crick base-pairing, 28

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