DEPARTMENT OF BIOCHEMISTRY

CHUKWUEMEKA ODUMEGWU OJUKWU UNIVERSITY

SEMINAR PRESENTATION

(BCH 491)

TOPIC APTAMERS AS THERAPEUTIC BIOMARKERS

PRESENTER OBI PRAISE ONYINYE

REGISTRATION NUMBER 2020 204 102

SUPERVISOR PROF LUKONG. C. BANBOYE

DATE

ABSTRACT

Aptamers are single strand DNA or RNA molecules, selected by an iterative process known as
Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Due to various
advantages of aptamers such as high temperature stability, animal free, cost effective production
and its high affinity and selectivity for its target make them attractive alternatives to monoclonal
antibody for use in diagnostic and therapeutic purposes. Aptamer has been generated against
vesicular endothelial growth factor 165 involved in age related macular degeneracy. Macugen
was the first FDA approved aptamer based drug that was commercialized. Later other aptamers
were also developed against blood clotting proteins, cancer proteins, antibody E, agents involved
in diabetes nephropathy, autoantibodies involved in autoimmune disorders, etc. Aptamers have
also been developed against viruses and could work with other antiviral agents in treating
infections. Biomarkers are detectable molecules that can reflect specific physiological states of
cells, organs, and organisms and therefore be regarded as indicators for specific diseases. And
the discovery of biomarkers plays an essential role in cancer management from the initial
diagnosis to the final treatment regime. Practically, reliable clinical biomarkers are still limited,
restricted by the suboptimal methods in biomarker discovery. Nucleic acid aptamers nowadays
could be used as a powerful tool in the discovery of protein biomarkers. Nucleic acid aptamers
are single-strand oligonucleotides that can specifically bind to various targets with high affinity.
As artificial ssDNA or RNA, aptamers possess unique advantages compared to conventional
antibodies. They can be flexible in design, low immunogenicity, relative chemical/thermos
stability, as well as modifying convenience.

Several SELEX (Systematic Evolution of Ligands by Exponential Enrichment) based methods

have been generated recently to construct aptamers for discovering new biomarkers in different
cell locations. Secretome SELEX-based aptamers selection can facilitate the identification of
secreted protein biomarkers. The aptamers developed by cell-SELEX can be used to unveil those
biomarkers presented on the cell surface. The aptamers from tissue-SELEX could target
intracellular biomarkers. And as a multiplexed protein biomarker detection technology, aptamer-
based SOMAScan can analyze thousands of proteins in a single run. In this review, we will
introduce the principle and workflow of variations of SELEX-based methods, including
secretome SELEX, ADAPT, Cell-SELEX and tissue SELEX. Another powerful proteome
analyzing tool, SOMAScan, will also be covered.
TABLE OF CONTENTS: APTAMERS AS THERAPEUTIC BIOMARKERS

REG NO: 2020204102

CHAPTER ONE

INTRODUCTION

1.1 General Introduction

CHAPTER TWO

APTAMERS AND BIOMARKERS

2.1 Aptamers

2.1.1 Types of Aptamers

2.1.2 Production of Aptamers

2.2 Applications of Aptamers

2.2.1 Therapeutic Applications

2.2.2 Diagnostic Applications

2.2.3 Biosensing and Detection

2.2.4 Enviromental and Food Safety

2.4 Biomarkers

2.4.1 functions of Biomarkers

2.4.2 Applications of Biomarkers

CHAPTER THREE

APTAMERS AS THERAPEUTIC BIOMARKERS

3.1 Aptamers in Therapeutic

3.1.1 Aptamers in Immuno Therapy

3.1.2 Aptamers in disease monitoring

3.2 Aptamers and the Diseases they treat

3.3 Gintatop (AS1411)

3.3.1 Gintatop Mechanism in Cancer treatment

3.3.2 Targeting Mechanism of Gintatop in Cancer treatment

3.4 Challenges in Aptamers Applications

CHAPTER FOUR

CONCLUSION

REFERENCE
 CHAPTER ONE

1.1 INTRODUCTION

Aptamers are single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules
that bind to protein targets by folding into a three-dimensional conformation, similar to
antibodies. The word aptamer comes from the Latin aptus, meaning fit, and the
Greek meros, meaning part or region. Aptamers are most often isolated by a method called
systematic evolution of ligands by exponential enrichment (SELEX), first described 25 years
ago. Since then, one aptamer has received approval from the US Food and Drug Administration
(FDA) to treat macular degeneration of the eye, and numerous others are in preclinical or clinical
trials. This review covers a brief history of aptamers and SELEX, describes postselection
optimization providing pharmacokinetic flexibility, and includes antidote oligonucleotide
reversal of aptamers to regulate aptamer activity rapidly. Finally, this review summarizes a list of
therapeutic aptamer targets that are currently in preclinical or clinical studies and offers
optimism that the future potential of aptamer therapeutics remains bright. Aptamers have a much
lower molecular weight than antibodies, allowing them to reach target tissue much more
efficiently, and may be less toxic. Autoimmune pathology can be a significant limiting factor in
achieving sufficient doses of antibodies for a patient to fully benefit from therapy; however,
aptamers may reduce this risk. 4-1BB is a costimulatory receptor that enhances T-
cell proliferation and amplifies CD8 + T-cell responses. An aptamer targeted against 4-1BB was
conjugated to a VEGF-targeted aptamer in order to target the entire complex to the tumor stroma.
This aptamer showed efficacy in a preclinical glioma model system, with a higher therapeutic
index than was seen using 4-1BB aptamer alone or a 4-1BB antibody (Schrand et al., 2014).
Aptamers targeted against other glioblastoma-associated antigens are actively being investigated
(Camorani et al., 2014) and are an emerging immunotherapeutic strategy that may be an
attractive alternative to antibodies. The use of biomarkers in basic and clinical research as well as
in clinical practice has become so commonplace that their presence as primary endpoints in
clinical trials is now accepted almost without question. In the case of specific biomarkers that
have been well characterized and repeatedly shown to correctly predict relevant clinical
outcomes across a variety of treatments and populations, this use is entirely justified and
appropriate. In many cases, however, the “validity” of biomarkers is assumed where, in fact, it
should continue to be evaluated and reevaluated. This article will consider the current conceptual
status of biomarkers as clinical and diagnostic tools and as surrogate endpoints in clinical
research with the goal of providing context for interpreting studies that rely heavily on such
biological measures.

CHAPTER TWO

2.1 APTAMERS

are short sequence of artificial DNA, RNA, XNA or peptide that binds a specific target molecule,
or family of target molecules. They exhibit a range of affinities (KD in the pM to μM range),[1]
[2] with variable levels of off- target binding[3] and are sometimes classified as chemical
antibodies. Aptamers and antibodies can be used in many of the same applications, but the
nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different
from amino acid -based structure of antibodies, which are protein. This difference can make
aptamers a better choice than antibodies for some purposes.
Left: Unbound aptamer. Right: the aptamer bound to its target protein. The protein is in yellow.
Parts of the aptamer that change shape when it binds its target are in blue, while the unchanging
parts are in orange. The parts of the aptamer that contact the protein are highlighted in red.

Most aptamers originate from SELEX, a family of test tube experiments for finding useful
aptamers in a massive pool of different DNA sequences. This process is much like natural
selection, directed evolution or artificial selection. In SELEX, the researcher repeatedly selects
for the best aptamers from a starting DNA library made of about a quadrillion different random
generated pieces of DNA or RNA . After SELEX, the researcher might mutate or change the
chemistry of the aptamers and do another selection, or might use rational design processes to
engineer improvements. for discovering aptamers also exist.
Researchers optimize aptamers to achieve a variety of beneficial features. The most important
feature is specific and sensitive binding to the chosen target. When aptamers are exposed to
bodily fluids, as in serum tests or aptamer therapeutics, it is often important for them to resist
digestion by DNA- and RNA-destroying proteins. Therapeutic aptamers often must be modified
to clear slowly from the body. Aptamers that change their shape drastically when they bind their
target are useful as molecular switches to turn a sensor on and off. Some aptamers are engineered
to fit into a biosensor or in a test of a biological sample. It can be useful in some cases for the
aptamer to accomplish a pre-defined level or speed of binding. As the yield of the synthesis used
to produce known aptamers shrinks quickly for longer sequences,[4] researchers often truncate
aptamers to the minimal binding sequence to reduce the production cost.

2.1.1 TYPES OF APTAMERS

DNA APTAMERS: DNA aptamers are single-stranded DNA oligonucleotide sequences that
bind to specific targets with high affinity. Currently, DNA aptamers can be produced only by in
vitro synthesis. It is difficult for DNA aptamers to have a sustained impact on intracellular
protein activity, which limits their clinical application. In this study, we developed a DNA
aptamer expression system to generate DNA aptamers with functional activity in mammalian
cells by mimicking retroviruses. Using this system, DNA aptamers targeting intracellular Ras
(Ra1) and membrane-bound CD71 (XQ2) were successfully generated in cells. In particular, the
expressed Ra1 not only specifically bound to the intracellular Ras protein but also inhibited the
phosphorylation of downstream ERK1/2 and AKT. Furthermore, by inserting the DNA aptamer
expression system for Ra1 into a lentivirus vector, the system can be delivered into cells and
stably produce Ra1 over time, resulting in the inhibition of lung cancer cell proliferation.
Therefore, our study provides a novel strategy for the intracellular generation of DNA aptamers
with functional activity and opens a new avenue for the clinical application of intracellular DNA
aptamers in disease treatment.

RNA APTAMERS: RNA Aptamers refer to RNA oligonulceotides that are capable of
binding to specific targets with high affinity and specificity. Through a process called Systematic
Evolution of Ligands by EXponential enrichment (SELEX), a number of RNA aptamers have
been identified against various targets including organic compounds, nucleotides, proteins and
even whole cells and organisms. RNA aptamers have proven to be of high therapeutic and
diagnostic value with recent FDA approval of the first aptamer drug and additional ones in the
clinical pipelines. It has also been found to be a particularly useful tool for cell-type specific
delivery of other RNA therapeutics like siRNA. All these establish RNA aptamers as one of the
pivotal tools of the emerging RNA nanotechnology field in the fight against human diseases
including cancer, viral infections and other diseases. This article summarizes the current
advancement in the identification of RNA aptamers and also provides some examples of their
therapeutic and diagnostic applications.

2.1.2 Production of APTAMERS

Aptamers consist of nucleic acids that bind to diverse targets with high affinity and selectivity.
They are screened through an in vitro process and usually have higher binding affinity than
traditional antibody. Aptamers are produced chemically, and no or little batch-to-batch variation
is observed during aptamer production. To describe molecular recognition properties for what
were nucleic acid-based ligands, they coined the term ‘aptamer’ using the Latin word “aptus”,
meaning “fitting” and the Greek word ‘‘meros’’, meaning “particle”.

SELEX is an in vitro selection method designed to identify aptamers that are selectively bound
to target molecules with high affinity. Substantive studies on aptamers have progressed since
the in vitro selection process called SELEX was first reported by Gold’s and Szostak’s
groups (Ellington and Szostak, 1990; Tuerk and Gold, 1990). First, the nucleic acid library,
which consists of 1014-1015 random oligonucleotide strands, is incubated with a target molecule.
Then, the target-bound oligonucleotide strands are separated from the unbound strands. The
target-bound DNA or RNA strands are eluted from the target molecule and amplified via
polymerase chain reaction to seed a new pool of nucleic acids. This selection process is
continued for 6-15 rounds with increasingly stringent conditions, which ensure that the nucleic
acid obtained has the highest affinity to the target molecule (Fig. 1). SELEX method can be
modified in a variety of ways to increase the specificity of aptamer and efficiency of SELEX.
Fig. 1.

Overview of SELEX scheme. Aptamers can be obtained through an iterative selection

process known as SELEX (systematic evolution of ligands by exponential enrichment) by
using single-stranded DNA or RNA. An initial pool of 10 14-1015 random oligonucleotide
(ONT) strands are subjected to binding with the target. Unbound ONTs are discarded and
RT-PCR or PCR is performed to amplify the targetbound ONTs. This selection process is
repeated 6-15 times using amplified ONTs as a new pool. This way, aptamers having high
specificity and affinity are screened. Diverse molecules can be the target of the SELEX,
including metal ion, protein, organic compound and cell. Toggle-SELEX performs SELEX
with two different target molecules to obtain bispecific aptamers.
Counter-SELEX

The counter-SELEX method was introduced to increase the efficiency of aptamer selection by
traditional SELEX (Fig. 1) (Jenison et al., 1994). Compared to traditional SELEX, counter-
SELEX has a pre-clearing step using closely related structural analogs of the target to effectively
discard non-specific aptamers. This allows dramatic improvement in aptamer selection and can
also be applied to other modified SELEX methods.

Cell-SELEX

The SELEX target is not limited to an individual molecule. The cell-SELEX strategy has been
described that utilizes living cells as the SELEX target (Fig. 1). Although traditional SELEX is
typically carried out using purified target molecules, whole live cells are also employed as
selection targets. Not only normal/abnormal mammalian cells such as virus-infected and cancer
cells but also live pathogenic organisms such as bacteria and viruses have been utilized as cell-
SELEX targets (Tang et al., 2009; Hamula et al., 2011). The aptamers generated are functional
with an original conformation of the target molecule on live cells. Compared to aptamers
selected using a purified target, a cell-SELEX-derived aptamer has more possibility to be used
directly for in vivo and clinical applications. A screened aptamer resulting from cell-SELEX
using abnormal cells can be used to detect disease or cancer. Moreover, biomarkers can be used
to identify the aptamer target for a specific abnormality (Blank et al., 2001). Many strategies are
available to select an anti-virus aptamer. First, SELEX can be performed using the virion
directly (Wang et al., 2000). Second, aptamers can be generated that specifically bind to virus-
infected cells. Through virus infection, viral proteins exist on the host cell surface due to the viral
gene or enveloped protein. SELEX provides screening of virus-specific aptamers that recognize
virus infected cells. Furthermore, aptamers can be selected for host cell proteins if their

expression levels are upregulated upon viral infection. Lastly, stable cell lines can be generated
that express viral protein as a cell-SELEX target, and the aptamer obtained can be used to detect
virus infected cells (Chen et al., 2009). Furthermore, novel biomarkers can be discovered using
the cell-SELEX technique. Because the target molecules of the aptamers generated by cell-
SELEX may be previously unrecognized as cell-specific surface molecules, they could be novel
biomarkers. Thus, cell-SELEX can be applied for de novo discovery of novel biomarkers for a
desired cell by identifying the aptamer binding partner. The cell- SELEX concept can be
extended for in vivo selection, which was first designed using a hepatic tumor xenograft mouse
model (Mi et al., 2010). Oligonucleotides were injected intravenously, liver tumors were
harvested, and the injected RNA molecules were extracted and amplified. A tissue-specific
aptamer can be more easily screened through this in vivo selection process. So, a screened
aptamer may be a useful target for a tissue of interest without non-specific biodistribution in
the in vivo application.

Capillary Electrophoresis-SELEX

The SELEX process has disadvantages in that it is time consuming to repeat the rounds. Some
molecular biological methods have been introduced to SELEX to overcome these disadvantages.
Capillary electrophoresis-SELEX (CE-SELEX) was designed for selecting aptamers to reduce
repeating rounds with low dissociation constants (Mosing et al., 2005). In this method, the
nucleic acids that bind the target migrate with different mobilities than those of unbound
sequences, allowing them to be collected as separate fractions. Although traditional SELEX
requires 6-12 rounds, CE-SELEX significantly reduces the number of rounds and increases
binding affinity (Schneider et al., 1995). CE-SELEX decreases the time, effort, and cost to
screen a higher affinity aptamer compared to those of traditional SELEX. Based on CE
technology, a non- SELEX method that selects an aptamer without amplification was
demonstrated (Berezovski et al., 2006). Non-equilibrium capillary electrophoresis of equilibrium
mixtures (NECEEM), a highly efficient affinity method, has been used to partition the DNA-
target complex from the free DNA. An aptamer with nanomolar affinity for the protein
farnesyltransferase was selected in a single round of partitioning using NECEEM (Berezovski et
al., 2005). The advantage of non-SELEX is its speed and simplicity. NECEEM-based non-
SELEX selection took only 1 h in contrast to several days or several weeks required for a
traditional SELEX procedure by conventional partitioning methods. This procedure can be
automated using a single commercially available capillary electrophoresis instrument.
One step MonoLEX

The MonoLEX approach combines a single affinity chromatography step with subsequent
physical segmentation of the affinity resin and one single final exponential amplification step of
the bound aptamers (Nitsche et al., 2007). Using the Biomek 2000, an automatic SELEX
device (Cox et al., 1998), Cox et al. demonstrated that 10 rounds of selection against eight
targets in parallel can be performed in 4-5 days.

Microfluidic SELEX

Microfluidic SELEX (mSELEX) was designed that combines traditional SELEX with a
microfluidic system. Protein immobilization is the most important aspect of mSELEX. A
mSELEX prototype device was developed and biotinylated lysozymes were immobilized to
silica microline previously coated with streptavidin (Hybarger et al., 2006). In another study, a
magnetic bead-based SELEX process with microfluidics technology and a continuous-flow
magnetic activated chipbased separation device was developed (Lou et al., 2009). Using this
mSELEX method, an enriched aptamer pool was obtained that tightly bound to the light chain of
recombinant Botulinum neurotoxin type A after a single round of selection with a 33 nM Kd
value. Recently, a novel formulation of solgel protein microarray material was developed, which
elicited physical properties suitable for protein immobilization, proteinprotein interactions, and
immunoassays (Kim et al., 2006). Based on sol-gel, the authors developed a microfluidic device
and method for binding nucleic acids to immobilized proteins and efficiently eluting and
recovering the intact nucleic acids (Park et al., 2009). The mSELEX system enhanced selection
efficiency and reduced time and effort compared to those of traditional SELEX.

Toggle-SELEX

Furthermore, toggle-SELEX has been established for in vitro animal model assays using a single
aptamer (Fig. 1). The “toggle” selection process was repeated during SELEX rounds using a
target applied to human thrombin for even rounds and porcine thrombin for odd rounds to select
a species cross-reactive aptamer (White et al., 2001). A species cross-reactive aptamer from the
toggle-SELEX strategy may contribute directly to animal models and clinical applications. Thus,
toggle-SELEX makes the aptamer technique closer to a “bench-to-clinic” concept.

Based on these novel methods, high-throughput screening of aptamers and improved efficiency
of the SELEX process is possible. Moreover, consilience technology such as microfluidic
technology and engineering could reduce effort, time, and cost of aptamer selection and analysis,
while increasing its efficacy. Taken together, aptamers may play a key role in future theragnosis
because of higher binding affinity, lower cost, and ease of synthesis compared to antibodies.

2.2 APPLICATION OF APTAMERS

Analogically to monoclonal antibodies, aptamers can specifically recognize and bind to their
target [73]. Therefore, following their isolation, aptamers can be utilized for molecular
recognition of their targets. Consequentially, aptamers have a number of diagnostic and
therapeutic applications, such as biosensors and target inhibitors. Due to simple preparation, easy
modification, and stability, aptamers have been used in the diverse areas within molecular
biology, biotechnology, and biomedicine.

2.2.1 THERAPEUTIC APPLICATION OF APTAMERS

Due to their ability to compete with small molecules and protein ligands and to inhibit their
targets [144], aptamers are considered to be promising therapeutics. Furthermore, aptamers can
activate the function of the target receptors or act as carriers for the delivery of therapeutic agents
to the target cells or tissue [9]. For example, aptamers have the potential to act as antiviral
agents. Previous work suggests that RNA aptamers against a synthetic derivative of gp120 can
neutralize HIV-1 [145]. Furthermore, RNA aptamers against RIG-I can inhibit Newcastle disease
virus (NDV), vesicular stomatitis virus (VSV), and influenza virus replication [144]. RNA
aptamers able to efficiently inhibit protease and helicase activities were developed which target
hepatitis C virus (HCV) NS3 helicase domain, thus inhibiting HCV [146,147,148]. Other
aptamers in discovery and preclinical stages include those targeting cancer, such as those binding
different growth factors (e.g. VEGF, bFGF, PDGF, KGF), NOX-A12 RNA aptamer binding the
chemokine ligand CXCL12 [149] and NAS-24 DNA aptamer targeting vimentin involved in
maintaining cell shape, cytoplasm integrity, and cytoskeleton stability [150]. Furthermore,
aptamers in discovery and preclinical stages include those against bacterial infections, such as
those caused by E. coli, S. aureus, M. tuberculosis, and Salmonella spp. [151,152]. A number of
aptamers for immune disorders are in the preclinical stages too, including ssDNA aptamer
BC007 targeting beta1-adrenoreceptor autoantibodies [153]. Furthermore, aptamers in the
preclinical stages of development include those targeting neurodegenerative diseases, immune
disorders, and inflammation [151].

A number of aptamers have entered clinical trials, such as for ocular diseases [154],
haematologic diseases [155], and cancer [156]. Pegaptanib, a vascular endothelial growth factor
(VEGF)-specific aptamer, was approved for therapeutic use for age-related macular
degeneration. Pegaptanib blocks VEGF which plays a key role in pathological angiogenesis, for
example, in ocular neovascular diseases, such as age-related macular degeneration (AMD) and
diabetic macular oedema [157]. AS1411, a G-rich DNA aptamer against nucleolin, is undergoing
phase II clinical trial for acute myeloid leukemia. The anti-tumor activity of AS1411 stems from
its ability to bind cell surface nucleolin, thereby inhibiting DNA synthesis, preventing cell
growth signaling, and inducing apoptosis [156]. AS1411 was shown to be efficient against a
variety of cancer cells, including lung cancer [156], colorectal cancer [158], breast cancer [159],
and hepatocellular carcinoma [160]. NOX-E36, l-RNA aptamer with 3′-PEG against chemokine
(C-C motif) ligand 2, is in phase I clinical trial for Type 2 diabetes and diabetic nephropathy.
NOX-E36 binds and neutralizes human chemokine CCL2 and related chemokines. This prevents
infiltration of pro-inflammatory cells into the kidney and allows resolving of the existing
inflammation over time [161,162].

2.2.2 DIAGNOSTIC APTAMERS

The high affinity and specificity of aptamers make them ideal diagnostic agents with the
potential to replace conventional antibodies in clinical diagnosis, environmental protection, and
food safety. Like monoclonal antibodies, aptamers can be used for the molecular recognition of
their respective targets. Aptamers have been successfully used for pathogen recognition, cancer
recognition, monitoring environmental contamination, and as stem cell markers.
Pathogen Recognition

The fluorescence resonance energy transfer (FRET)-aptamers were developed as a novel high-
throughput screening tool against Escherichia coli outer membrane proteins to detect
enterotoxaemia E. coli (ETEC) K88 [74]. Furthermore, aptamers were utilized to detect surface
proteins of Campylobacter jejuni [75]. In addition to using purified bacterial proteins as targets,
the whole bacterium-based SELEX procedure was applied to detect E. coli [76], Lactobacillus
acidophilus [76], Staphylococcus aureus [77], the virulent strain of Mycobacterium
tuberculosis [78], Vibrio parahemolyticus [79], Shigella sonnei [78], and C. jejuni [80]. This led
to development of aptamers with increased affinity and specificity. SELEX-based approaches
can be also used to generate molecular probes for detecting viral infections, such as vaccinia
virus [81], herpes simplex virus [82], hepatitis C virus [83,84], hepatitis B virus [83,84], human
immunodeficiency virus [85], influenza virus [86], and Severe Acute Respiratory Syndrome
(SARS) coronavirus [87]. Furthermore, SELEX has been used successfully to generate aptamers
for the detection of a number of parasites, such as Trypanosoma spp. [88], Leishmania spp.
[89], Plasmodium spp. [90,91,92,93,94,95,96,97,98,99], Cryptosporidium
parvum [100], Entamoeba histolytica [101].

Cancer Recognition

Development of aptamers for a reliable and timely cancer diagnosis and prognosis evaluation is
of the highest importance. To address this issue, aptamers have been developed for the detection
of a number of cancer-related biomarkers [102], including multiple tumor-related proteins in
living cancer cells, such as MUC1 (mucin 1), HER2 (human epidermal growth factor receptor 2),
and estrogen receptor [102]. Aptamers for the detection of the MCF-7 breast cancer cells [103]
and leukemia CCRF-CEM cells were also developed recently [104]. In addition, aptamers have
been successfully used for the detection of a number of tumor-related soluble biomarkers,
including carcinoembryonic antigen (CEA), prostate specific antigen (PSA) [105,106].
Fluorescently labeled aptamers showed high detection rates of the metastatic tumor tissues [107].
Furthermore, aptamers were successfully used also for the in vivo imaging of lymphoma,
adenocarcinoma, leukemia, glioblastoma and other cancer types [102,108]
Monitoring Environmental Contamination

Aptamers have the potential to monitor and to minimize the environmental pollutants and the
resulting illnesses. Antibiotics, heavy metals, toxins, and pathogens can be toxic to nervous,
endocrine, and reproduction systems. Antibiotics used for farm animals may accumulate in the
animal tissues and transmit to human upon ingestion. To address this issue, aptamers have been
developed against some antibiotics, such as chloramphenicol [112] and tetracycline [113].

Furthermore, aptamers for a number of environmental toxins have been developed recently.
These include aptamers against ochratoxin A (OTA) [114], bacterial endotoxins [115], and
bisphenol A [116]. Aptamers against mercury [117,118], arsenic [119,120,121], copper [122],
and lead [123] have also been generated to identify heavy metal contamination.

Aptamers for the detection of various pesticides in the environment have been also developed
recently, such as fungicide carbendazim [124], acetamiprid and atrazine [125,126], and
chlorpyriphos [127].

In addition, aptamers have been generated against various types of herbicides [128] and
insecticides [129], which may cause reproductive damage in humans. Abraham et al. reported on
the identification and characterization of an ssDNA aptamer against herbicide atrazine, recently
[130].

2.2.3 BIOSENSING AND DETECTION

The ease of which different aptamer structures can be generated together with their ability to
bind specific targets by forming stable tertiary structures has led to widespread applications of
aptamers in biosensors [123,131,132,133].Besides monitoring environmental contamination
described above, examples of the recently developed biosensors include the following:
(1) Aptamer-based biosensor to target disease biomarkers, such as platelet-derived growth factor
BB (PDGF-BB), a cancer related protein, to help early diagnosis and prognosis of cancer
development [134].

(2) Biosensors for the azole class of antifungal drugs with application in the therapeutic drug
monitoring of patients with invasive fungal infections [135].

(3) Biosensors harboring DNA aptamers targeting B-lactoglobulin for the detection of milk
allergen [124].

(4) Highly sensitive biosensors for the detection of human epidermal growth factor receptor 2
with application in real-time detection of breast cancer cells [136].

(5) Biosensors for the detection of bisphenol A [137].

A good example of this is the graphene-based biosensor targeting B-globulin for the detection of
milk allergen [124] and the biosensor for the detection of human epidermal growth factor
receptor 2 [136] described above. In the latter, ultrafine graphene nanomesh, a continuous 2D
graphene nanostructure with a high density of holes punched in the basal plane, has been created
to improve the signal on/off ratio [136]. Biosensors can be further enhanced by using
biomaterials, such as antibodies to form a "sandwich" structure. In the biosensor developed by
Wiedman et al. for the azole class of antifungal drugs, two aptamers were combined to form a
"sandwich" structure [135].

Currently, change in the biosensor’s electric signal (current and resistance) upon binding to
analytes is the major principle of detection, while the electrochemical impedance spectroscopy
(EIS) and cyclic voltammetry (CV) are usually used to monitor aptamer–ligand complexes
occurring on electrode surface [133]. DNA-based electrochemical biosensors have been widely
used for the detection of heavy metal ions, including mercury, lead, and copper [138].
Fluorescence-labeled aptamer probes have also been widely used in biosensors (Figure 3). A
novel label-free aptasensor for acetamiprid through fluorescence resonance energy transfer
(FRET) involves the conformational change taking place after the aptamer binds to acetamiprid
and its effect on the stability of the gold nanoparticles in solution. In this biosensor, dual-colored
Au NCs (gold nanoclusters) excitable by single-wavelength excitation were used as energy
donors to achieve simultaneous detection of multiple tumor markers [139]. Another recent
example of the fluorescent biosensor is the biosensor for the detection of bisphenol A based on
the adsorption ability of a nano-sized iron metal–organic framework (Fe-MIL-88B–NH2) to a
DNA aptamer [137].

To further enhance the affinity of detection, recent studies have focused on three aspects:
reconstructing aptamers, modifying electrodes and exploiting the inter-substance interactions. It
has been demonstrated that removing the non-binding domain of aptamers could increase the
binding affinity and specificity of the aptamer-target complex by improving the density of the
binding domains [140].

Furthermore, the rolling circle amplification increases the length of the aptamer, thus increasing
the number of binding domains. Electrochemical roughening approach also enhanced the
signaling of electrochemical sensors by increasing the microscopic surface area of gold
electrodes [141]. This simple method does not require pretreatment compared to the
conventional electrochemical deposition to increase the electrode surface [141]. Signal
amplification was also achieved via synergetic catalysis using DNAzyme-decorated AuPd
nanoparticles. The signal was amplified by synergistic catalysis of
G-quadruplex/hemin/HRP/AuPd/poly(o-phenylenediamine) bioconjugates, thus greatly
enhancing the sensitivity of the biosensor [142]. Jeddi and Saiz presented the first approach to
predict three-dimensional structures of aptamers from sequences by focusing explicitly on
ssDNA hairpins [143]. Different 3D aptamer configurations resulted in different affinities, thus
highlighting the role of 3D configuration of aptamers in their sensitivity, specificity, and
reliability.

2.2.4 ENVIRONMENT AND FOOD SAFETY

Food maybe contaminated by chemical compounds, toxin or pathogen leading to various food
borne ailments. According to World Health Organization (WHO) food borne and waterborne
diarrhoeal diseases kill about 2.2 million people annually out of which 1.9 million are children
[38]. This growing public health problem demands the development of highly sensitive
technologies for rapid detection of food contaminants. In this context, biosensors have a potential
to emerge as a major tool in maintaining food safety. Traditional techniques are largely based on
antibody-conjugated biosensors. However, many food contaminants are not conducive for
antibody generation. Also antibodies suffer from batch to batch variation and short shelf lives
necessitating the development of a simpler, consistent and cost effective approach for food safety
testing.
Aptamers can address many of the challenges of a traditional biosensor without compromising
on specificity and affinity. Based on the type of target, aptamers used in food safety testing and
environmental pollution control can be broadly classified under two groups-aptamers against
small molecule contaminants and those against pathogens.

2.4 BIOMARKERS

The term “biomarker”, a portmanteau of “biological marker”, refers to a broad subcategory of

medical signs – that is, objective indications of medical state observed from outside the patient –
which can be measured accurately and reproducibly. Medical signs stand in contrast to medical
symptoms, which are limited to those indications of health or illness perceived by patients
themselves. There are several more precise definitions of biomarkers in the literature, and they
fortunately overlap considerably. In 1998, the National Institutes of Health Biomarkers
Definitions Working Group defined a biomarker as “a characteristic that is objectively measured
and evaluated as an indicator of normal biological processes, pathogenic processes, or
pharmacological pharmacologic responses to a therapeutic intervention.” [**1] A joint venture
on chemical safety, the International Programme on Chemical Safety, led by the World Health
Organization (WHO) and in coordination with the United Nations and the International Labor
Organization, has defined a biomarker as “any substance, structure, or process that can be
measured in the body or its products and influence or predict the incidence of outcome or
disease” [2]. An even broader definition takes into account not just incidence and outcome of
disease, but also the effects of treatments, interventions, and even unintended environmental
exposure, such as to chemicals or nutrients. In their report on the validity of biomarkers in
environment risk assessment, the WHO has stated that a true definition of biomarkers includes
“almost any measurement reflecting an interaction between a biological system and a potential
hazard, which may be chemical, physical, or biological. The measured response may be
functional and physiological, biochemical at the cellular level, or a molecular interaction.” [3].
Examples of biomarkers include everything from pulse and blood pressure through basic
chemistries to more complex laboratory tests of blood and other tissues. Medical signs have a
long history of use in clinical practice—as old as medical practice itself—and biomarkers are
merely the most objective, quantifiable medical signs modern laboratory science allows us to
measure reproducibly. The use of biomarkers, and in particular laboratory-measured biomarkers,
in clinical research is somewhat newer, and the best approaches to this practice are still being
developed and refined. The key issue at hand is determining the relationship between any given
measurable biomarker and relevant clinical endpoints.

2.4.1 FUNCTIONS OF BIOMARKERS

1. Biomarkers as surrogate endpoints: When used as outcomes in clinical trials, biomarkers

are considered to be surrogate endpoints; that is, they act as surrogates or substitutes for
clinically meaningful endpoints. But, not all biomarkers are surrogate endpoints, nor are they all
intended to be. Surrogate endpoints are a small subset of well-characterized biomarkers with
well-evaluated clinical relerelevance [**1]. To be considered a surrogate endpoint, there must be
solid scientific evidence (e.g., epidemiological, therapeutic, and/or pathophysiological) that a
biomarker consistently and accurately predicts a clinical outcome, either a benefit or harm. In
this sense, a surrogate endpoint is a biomarker that can be trusted to serve as a stand-in for, but
not as a replacement of, a clinical endpoint.

Even biomarkers that are statistically validated to be surrogates for a given clinical endpoint may
not actually be part of the pathophysiological pathway that results in that endpoint. In some
cases, there may be evidence that the biomarkers measure a process or product of a key pathway
stage, but assuming this relationship in all cases risks mistaking correlation for causation. Many
possible explanations exist for biomarkers that correlate with clinical endpoints under only
limited circumstances. For example, multiple interrelated disease pathways may be involved, or
the biomarkers might be indirect signs of a pathway that are not fundamental to the key disease
processes.

There are a number of advantages to using biomarkers as surrogate endpoints in trials. Primary
clinical endpoints, such as survival, can occur so infrequently that their use in clinical trials can
be highly impractical, or even unethical. For many diseases, clear clinical endpoints such as
survival or recurrence of, for instance, a cardiovascular event may occur only after many years of
treatment. Biomarkers can provide researchers interim evidence about the safety and efficacy of
such treatments while more definitive clinical data is collected. In some cases, it may be
preferable to use established biomarkers as surrogate endpoints to reduce the risk of harm to
subjects: the early data provided by biomarkers can allow researchers the opportunity to stop
interventions potentially harmful to subjects before the associated clinical data would be
available. In other cases, biomarkers may simply allow researchers to design smaller, more
efficient studies, reducing the number of subjects exposed to a given experimental treatment. By
shortening the time to approval of new treatments, more efficient trials could speed the overall
drug development process, allowing effective treatments to reach their target patient populations
sooner, while conserving both material and human resources for other research projects, even
other clinical trials.

Of note, the term “surrogate marker” – in contrast to the term “surrogate endpoint” – has been
criticized for inaccurately suggesting the surrogate, in this case a biomarker, is a stand-in for
another marker [**1]; in fact, biomarkers as surrogate endpoints should be stand-ins only for
clear clinical endpoints, and their use as surrogate endpoints is only appropriate under limited
circumstances.

2. Biomarkers as provisional always: Biomarkers have been approved by the U.S Food
and Drug Administration (FDA) regulation for use as surrogate endpoints in the treatment
dedevelopment process [8]. The FDA allows provisional intervention approval with surrogate
marker-defined efficacy but further requires phase IV follow-up studies that prove relevant
clinical endpoint correlation exists. Some cautious researchers and commentators have suggested
that biomarkers are most effective in and best left for use as endpoints in phase I and phase II
trials. Their use can help determine what potential treatments are worth the effort and resources
of a large, well-powered phase III trial. There is, of course, the risk that researchers may
inappropriately abandon large-scale research on treatments that, although actually effective at
improving clinical outcomes, do not appear to be effective on the basis of biomarker analysis.
This type of false negative has in fact already occurred in at least one case involving a trial of
chronic granulomatous disease in children [9]. The trial, which was designed to measure both
surrogate outcomes and true clinical endpoints, ultimately showed that the treatment, interferon
gamma, was effective at reducing mortality, but there was no associated improvement on the
surrogate outcome, production of superoxide, which was expected to increase the patients' ability
to kill bacteria. The biological process that led to improved clinical outcomes, in other words,
was not captured by the biological mechanism proposed and predicted by the researchers. Had
the trial been designed to rely first on interim analysis of the biomarkers alone, the true and
clinically relevant effects of the treatment might never have been discovered. The lesson here is
the same as in the cases of false positives: treatments' effects on biomarkers used as surrogate
outcomes do not necessarily predict true clinical outcomes. Without confirmatory clinical
endpoint analysis, the overreliance on biomarkers, even ones previously considered validated in
particular treatment contexts, presents a serious and persistent risk of producing misleading, and
in some cases dangerous, erroneous conclusions.

2.4.2Applications of Biomarkers

Diagnostic biomarkers

A diagnostic biomarker detects or confirms the presence of a disease or condition of interest, or

identifies an individual with a subtype of the disease.3 As we move into the era of precision
medicine, this type of biomarker will evolve considerably. Such biomarkers may be used not
only to identify people with a disease, but to redefine the classification of the disease. For
example, the detection of cancer is moving rapidly toward a molecular and imaging-based
classification rather than a largely organ-based classification scheme.
Given a diagnostic biomarker that can be measured with sufficient precision and reliability with
a delineated context of use, the assessment of that biomarker remains complex. One goal is to
define a method for validation that assures that the biomarker can be measured reliably,
precisely, and repeatably at a low cost. All too often, assays are not validated, engendering
misleading assumptions about the biomarker’s value. The complexity of validation can be seen
in the use of troponin, clearly an important biomarker for the diagnosis of acute myocardial
infarction. The operating characteristics of the many assays for troponin vary considerably,
especially at the lower limit threshold, where misclassification can lead to a major difference in
medical care. Furthermore, while the advent of high-sensitivity troponin assays has opened many
avenues for sophisticated diagnosis of small episodes of myocardial necrosis, it has created
further confusion in the field. When small elevations of troponin occur at previously
undetectable levels, the clinical consequences are unclear. We can expect that as measurement
methods continue to improve, the understanding of the value of individual diagnostic biomarkers
will likewise evolve.

If a diagnostic biomarker moves beyond a general application, such as advancing scientific

concepts, to specific use in prospective research or clinical practice, close attention must be paid
to the context of use. A diagnostic biomarker may be useful in one set of clinical circumstances
but completely misleading in another context. For example: in low-prevalence diseases such as
pancreatic or ovarian cancer for which a new diagnosis is psychologically devastating or would
require invasive evaluation, a biomarker must have a very low false-positive rate. On the other
hand, in screening for common diseases such as hypertension or hyperlipidemia for which
repeated assessments can be done with little risk, higher false-positive rates are tolerable and the
focus of concern may be on false-negative rates.

The use of receiver-operating characteristic curves has enabled a rational process of diagnostic
biomarker evaluation to proceed.5 A common problem, however, is the absence of a historical
standard for defining the presence or absence of the disease or condition. Furthermore, decision
thresholds and clinical utility are becoming important measures for assessing the value of
biomarkers for clinical application. In the future, proof that a biomarker adds information about
diagnosis may be necessary but not sufficient. Rather, the key question will be whether the
additional information is substantial enough to lead to a change in clinical decision-making.
Statistics for evaluating this issue, such as the net reclassification index, are evolving.6
Researchers involved in early preclinical biomarker research would be well served to understand
how the biomarker will eventually be evaluated, just as those doing early drug development
should have the ultimate use in humans in view.7

Monitoring biomarkers

When a biomarker can be measured serially to assess the status of a disease or medical condition
for evidence of exposure to a medical product or environmental agent, or to detect an effect of a
medical product or biological agent, it is a monitoring biomarker. Monitoring is a broad concept,
so there is overlap with other categories of biomarkers as described below.

Monitoring biomarkers have important applications in clinical care. When blood pressure is
treated or low-density lipoprotein (LDL) cholesterol-lowering drugs are used, blood pressure or
LDL cholesterol levels are monitored. Similarly, when HIV infection is treated, CD4 counts are
monitored. But while the general concept of monitoring for clinical purposes is intuitive, arriving
at a more refined understanding of what changes in the biomarker should signal a particular
change in clinical course and decision-making (e.g. more testing or intervention) is complex and
often less precise than is desirable. For example, target measurements for hemoglobin
(Hb)A1C,8 blood pressure,9 and LDL cholesterol10 remain controversial despite these being
among our most well-studied and accepted biomarkers. Similarly, we often lack sufficient
empirical confirmation of the most helpful interval between measurements or the duration of the
clinical course during which measurements should be made. Many biomarkers routinely used in
clinical practice have very imprecise operating characteristics, so that they are used in a clinical
“gestalt” along with the phrase “clinical judgment is needed.” Yet the specifics of clinical
parameters that should go into a good clinical judgment are unspecified.

When medical products are developed, changes in biomarkers are routinely used to make
decisions about whether key thresholds have been reached, allowing developers to conclude that
the therapy affected a biological target enough to merit continued development of the product.
Most initial biomarkers used for this purpose measure effect on the assumed target of the
intervention, so that changes in the biomarker indicate target engagement and related activity. As
discussed below, the ability to measure off-target effects on biological systems will increasingly
bring panels of biomarkers and systems measurement into play to evaluate intermediate findings
in medical product development.

Monitoring biomarkers are also important in ensuring the safety of human research participants.
For example, the safety threshold for drugs with possible liver toxicity is monitored through
serial measurement of liver function tests, and cardiovascular events are measured through the
use of serial troponins.

Monitoring biomarkers are also useful for measuring pharmacodynamic effects, to detect early
evidence of a therapeutic response, and to detect complications of a disease or therapy.
International normalized ratio (INR) is a classical pharmacodynamic measure used to titrate the
dose of warfarin anticoagulation. Similarly, when blood pressure is treated, a reduction in the
measure of blood pressure provides evidence that the therapy is working.

One of the more interesting aspects of monitoring biomarkers is the almost unalterable belief
held by many researchers and clinicians that changes in biomarker measurements give the best
measure of the likely outcome for a patient or population. However, in many circumstances the
actual measure, not the change, is the best predictor of outcome, even if the change is the best
way to monitor whether the therapy itself is having an effect. For example, an angiotensin-
converting enzyme (ACE) inhibitor may cause an elevation of serum creatinine and/or
potassium, and this provides a measure of drug effect. However, the risk to the patient or
research participant is primarily determined by the actual creatinine or potassium level, not the
change in levels.

Pharmacodynamic/response biomarkers

When the level of a biomarker changes in response to exposure to a medical product or an

environmental agent, it can be called a pharmacodynamic/response biomarker. This type of
biomarker is extraordinarily useful both in clinical practice and early therapeutic development. If
one is treating hypertension or diabetes and no reduction in blood pressure or glucose occurs
with a therapy, there is good reason to eschew that intervention and pursue another. Similarly, a
candidate drug for a condition that does not alter the key parameter of that biomarker in phase 1
trials would hardly be worth pursuing. A special circumstance is phase 1 studies of normal
individuals. It would be unexpected for a disease-related biomarker to show a major change (for
example, blood pressure) in persons with normal baseline values. In this circumstance, the main
focus is on developing preliminary evidence that the drug will be safe to use in individuals with
the target disease. For many drugs, dosing is determined by measured change in a
pharmacodynamic/response biomarker when a therapy is given.

However, the interpretation of pharmacodynamics/response biomarkers is not always simple or

straightforward. In the case of ACE inhibitors, the initial view was that acute titration of dose in
the intensive care unit could guide dosing in heart failure patients. And indeed, it was possible to
see major differences in the responsiveness of different patients to the same dose. But
unfortunately these acute responses did not adequately predict long-term responses. It is
therefore critically important to validate that the measured change in the
pharmacodynamics/response biomarker provides a reliable signal for the expected therapeutic
response.

Another complex problem arises when easily measureable biomarkers do not reflect true
pharmacodynamic responses. With intravenous fibrinolytic agents, serum pharmacokinetics do
not reflect the activity of the agent in the thrombus. Similarly, amiodarone is heavily deposited in
fat and therefore has a much longer duration of activity than simple measurement of serum levels
would predict.

Predictive biomarkers

A predictive biomarker is defined by the finding that the presence or change in the biomarker
predicts an individual or group of individuals more likely to experience a favorable or
unfavorable effect from the exposure to a medical product or environmental agent.3 Proving that
a biomarker is useful for this purpose requires a rigorous approach to clinical studies. Ideally,
patients with or without the biomarker are randomized to one of two or more treatments (or a
placebo comparator) and differences in outcome as function of treatment are significantly related
to the difference in presence, absence, or level of the biomarker. Proof of a reliable predictive
biomarker thus represents a “high hurdle” to clear.

Predictive biomarkers are important for enrichment strategies11,12 in the design and conduct of
clinical trials. Especially in the pre-registration phase of development, focusing enrollment on
participants with elevated levels of a predictive biomarker enables a clearer signal that the
treatment actually has an effect by enrolling people in whom the treatment is likely to “work.”
Using predictive biomarkers for enrichment is a more targeted approach than using prognostic
biomarkers, which can be used to increase event rates but not to select specific patients who are
more likely to respond or not respond to therapy.

The same thinking underlies much of the current consensus about treatment choice in clinical
practice. Antihypertensive medications are prescribed for patients with elevated blood pressure;
blood transfusion is used in people with anemia measured by low Hb levels; acute reperfusion is
indicated in patients with ST-segment elevation on an electrocardiogram—all of these are
examples of biomarkers that differentially select patients likely to respond to therapy. Similarly,
populations at increased risk due to high levels of predictive biomarkers are identified as needing
additional intervention in population health strategies. For example, patients with high levels of
HbA1C have the most to gain from aggressive therapies to treat diabetes. In addition, a major
growth area in predictive biomarkers is the development of genetic and genomic markers for
precision medicine, as in the case of cancer patients with HER2 receptor positive assays who are
more likely to respond to treatment with herceptin.

The biomarker-guided use of LDL cholesterol-lowering drugs offers an excellent example of the
complexity of these issues. LDL cholesterol is clearly a susceptibility/risk biomarker and a
prognostic biomarker. Patients with elevated LDL cholesterol are at increased risk both of
developing atherosclerosis and of experiencing an event such as death, stroke, or myocardial
infarction once they have diagnosed disease. Statins, the selective cholesterol absorption
inhibitor ezetimibe, and PCSK9 inhibitors all lower LDL cholesterol levels and reduce mortality
and critical clinical events such as stroke. However, in multiple clinical trials cumulatively
enrolling more than 100,000 patients, the relative effect on event reduction is similar across all
levels of LDL cholesterol, including levels well within the normal range.13 Therefore, in clinical
trials, the event reduction is a function of the overall relative risk reduction and the absolute risk
of an event, which is determined not only by LDL cholesterol levels, but also by multiple factors,
including age, smoking status, diabetes, and blood pressure. Environmental exposures have
similar characteristics. Individuals and subpopulations may have particular risks associated with
specific biomarkers such that preventive measures are most likely to be useful in people with
elevated levels of those biomarkers.

Prognostic biomarkers

A prognostic biomarker is used to identify the likelihood of a clinical event, disease recurrence,
or disease progression in patients with a disease or medical condition of interest. Although this
distinction is not uniformly accepted, the BEST working groups concluded that prognostic
biomarkers should be differentiated from susceptibility/risk biomarkers, which deal with
association with the transition from healthy state to disease. Furthermore, they are distinguished
from predictive biomarkers, which identify factors associated with the effect of intervention or
exposure.

In clinical trials, prognostic biomarkers are routinely used to set trial entry and exclusion criteria
to identify higher-risk populations. The key issue is that the statistical power of a trial is
determined by the number of events rather than the sample size. When trials are enriched in this
manner, the event rates are increased; if the treatment is effective, the differences in outcomes as
a function of treatment are magnified quantitatively but not qualitatively. In addition, prognostic
biomarkers are especially important for predicting the risk of an event or poor outcome in an
individual. This information is key to decisions about length of stay in hospital and/or in
intensive care units. Yet another major use of prognostic biomarkers is for resource allocation in
population health: by stratifying the risk for both negative clinical and financial outcomes, a
healthcare organization can distinguish which patients could benefit from more intensive
evaluation while allowing others to avoid unnecessary additional diagnostic tests or medical
interventions.

Safety

A safety biomarker is measured before or after an exposure to a medical intervention or

environmental agent to indicate the likelihood, presence, or extent of a toxicity as an adverse
event. For many therapies, monitoring for hepatic, renal, or cardiovascular toxicity is critical to
assuring that a given therapy can be safely sustained.

Safety biomarkers are useful for identifying patients who are experiencing adverse effects from a
treatment. When antiarrhythmic drugs are prescribed, prolongation of the QT interval on the
electrocardiogram is used as a safety biomarker because it predicts the risk of developing the
lethal arrhythmia torsades de pointes and can be used to identify patients in need of
countermeasures for effective therapy. Similarly, safety biomarkers can be used to monitor a
population for exposure to an environmental risk or to monitor a population after an exposure.

An interesting aspect of developing safety biomarkers is the balance that should be sought
between safety and the potential benefits of therapy. Returning to the example of QT interval
monitoring: the effect such monitoring has had on drug development has been a topic of frequent
discussion and controversy. It is possible that a drug whose benefits outweighed its risks has
been missed because development was stopped when QT interval prolongation was detected.
The Cardiac Safety Research Consortium, which includes representatives from the FDA,
industry, and academia, is working on strategies for establishing an optimal balance between the
ability to measure risk through early biomarker detection with the potential for benefit.14

Susceptibility/risk

A biomarker that indicates the potential for developing a disease or medical condition in an
individual who does not currently have clinically apparent disease or the medical condition is
classified as a susceptibility/risk biomarker. The concept is similar to prognostic biomarkers,
except that the key issue is the association with the development of a disease rather than
prognosis after one already has the diagnosis. These types of biomarkers are foundational for the
conduct of epidemiological studies about risk of disease.

CHAPTER THREE

APTAMERS AS THERAPEUTIC BIOMARKERS

3.1 Aptamers in Therapeutics

Aptamers can be used for therapeutic purposes in much the same way as monoclonal antibodies.
However, unlike traditional methods for producing monoclonal antibodies, no organisms are
required for the in vitro selection of oligonucleotides. This freedom from cellular biochemistry
offers a huge advantage in manipulating the process of directed evolution. As we will see,
chemistry, selection conditions and targets can be manipulated in vitro in ways that would be
difficult or impossible if organisms were involved.

In addition, aptamers have a unique niche relative to other oligonucleotide therapeutics.

For antisense oligonucleotides or siRNAs, the therapeutic target is intracellular, whereas aptamer
therapeutics can be developed for intracellular, extracellular or cell-surface targets. Targeting
proteins in these latter two classes alleviates the necessity for the therapeutic to cross the cell
membrane. Much like monoclonal antibodies13, aptamers can theoretically be used
therapeutically in any disease for which extracellular blockade of protein–protein interactions is
required. The focus on extracellular targets has so far not been a limiting factor for aptamer
development, as aptamers are currently undergoing clinical evaluation for ocular diseases,
haematological diseases and cancer14,15.

3.1.1 Aptamers in Immuno Therapy

Cancer immunotherapy is based on the observation that tumor cells vary from healthy cells
quantitatively and qualitatively in their protein (and potential antigen) repertoire. Cells of the
immune system can in principle recognize unique tumor markers as antigen and they are
therefore often referred to as tumor-associated antigen. In an ideal scenario, the generated
response would be sufficient to destroy the tumor and indeed this is sometimes the case in the
very early stages of tumor onset. Unfortunately, the strength of this type of response is usually
weak and worse yet, tumors develop mechanisms over time that lead to evasion of immune
recognition. These mechanisms include the expression of inhibitory ligands to induce down-
regulation of immune function through negative co-stimulation (Dong et al., 2002; Chen, 2004).

The modulation and particularly the enhancement of tumor immune responses is of great
therapeutic interest in the context of tumor immunotherapy. One approach has the potential to
block undesired immune down-regulation to enhance therapeutic efficacy; another is to engage
stimulatory signals. Antibodies, immunological receptors, cytokines, and chemokines have been
targeted primarily through the development of antibodies or soluble version of receptor ligands
or recombinant cytokines. Thus far, only few aptamers has been developed targeting immune
modulatory proteins. Most of these were developed to inhibit immune responses and intended for
application for the treatment of autoimmune diseases rather than the treatment of cancer by
enhancing anti-tumor immune responses.

Antibodies

The first immunological molecules for which aptamers were isolated were antibodies (Table 2).
Early work by Tsai, et al. targeted polyclonal antibodies raised in rabbits against a peptide
portion of the bacteriophage T7 gene 10 protein (g10) (Tsai et al., 1992). Though the antibodies
naturally interacted with g10, the isolated RNA antibodies were able to compete for antibody
binding. Similarly, the approach was also extended to sera obtained from patients with the
autoimmune disease systemic lupus erythematosus (SLE) (Tsai and Keene, 1993). This study
isolated aptamers that contained a motif found in the U1 small nuclear RNA and was able to
compete for autoantibody binding to U1 contained in the patient serum. These experiments were
among the first to demonstrate the capacity of SELEX to isolate RNAs to non-nucleotide binding
proteins and the first targeting immunological molecules. Additional studies published by
Doudna, et al. (Doudna et al., 1995) as well as Lee, et. al. (Lee and Sullenger, 1996) isolated
aptamers to monoclonal antibodies, including an antibody specific to the human insulin receptor,
which binds to a major immunogenetic epitope. The generated aptamers block the interaction of
the antibody with the insulin receptor and ameliorate the generated autoimmune response.
SELEX against a monoclonal myasthenic antibody also generated a decoy, capable of blocking
the interaction between the antibody antigen, the human acetylcholine receptor, and the antibody
(Lee and Sullenger, 1997; Hwang and Lee, 2002). This example concludes the list of aptamers
targeting the antigen binding sites of antibodies. An aptamer developed against human IgE,
however, was intended to block the interaction of the antibody to its receptor, Fcε Receptor I,
rather than its antigen. The aptamer blocks the pathologic interaction of overexpressed IgE with
the receptor in vitro, as is the case in allergic diseases such as allergies and asthma (Wiegand et
al., 1996). Furthermore, this aptamer has been used as detection probes in atomic force
microscopy (AFM) detection studies of IgE (Lin et al., 2006) as well as in microarrays (Cho et
al., 2006).
Cytokines

Cytokines and chemokines are proteins that affect the growth, proliferation and function of
immune effector cells of the innate and adaptive immune system. They play a vital role in the
regulation of the magnitude and duration of immune responses. This class of proteins can
function either in an agonistic or antagonistic manner.

IFNγ is a member of the interferon family of cytokines. This immune stimulatory cytokine is
secreted by activated lymphocytes, dendritic cells, and NK cells. Its function includes the
enhancement of immune cell activity leading to increased anti-tumor responses. The presence of
IFNγ can therefore be beneficial for the treatment of tumors (Ikeda et al., 2002). However, it has
also been involved in the development of autoimmune diseases (Kubik et al., 1997). An aptamer
developed to human IFNγ blocks binding of the cytokine to its receptor and could potentially be
used for the treatment of inflammatory diseases (Kubik et al., 1997)Oncostatin M is a member of
the interleukin 6 cytokine family. Just as IFNγ, this cytokine also has proinflammatory function
and can therefore enhance tumor immune responses but also aggravate autoimmune disease. The
developed aptamer agonist has demonstrated the ability to block oncostatin-M receptor binding
and downstream signaling event in vitro (Rhodes et al., 2000).

Transforming growth factor β1 and 2 (TGFβ1 & TGFβ2) can induce cell growth or apoptosis and
their inhibition has been shown to be useful for cancer therapy. Antagonistic aptamers to both
targets have been developed (McCauley et al., 2006; Kang et al., 2008), though their effect on in
vivo tumor growth remains to be determined.

Chemokines

Chemokines are agents that can induce cellular chemotaxis and thereby act as proinflammatory
agents . MCP-1 is an example of such a chemoattractant. An aptamer agonist can block
chemokine function and could reduce autoimmune responses in a therapeutic setting. Another
aptamer targeting a chemokine CXCL-10 (Rhodes et al., 2001) was developed to elucidate the
biological function CXCL-10 (Marro et al., 2005). The antagonistic aptamer can block CXCL10
induced signaling, which may or may not be useful in the treatment of cancer since studies have
shown that CXCL-10 can take on juxtaposing roles in cancer development as it can promote as
well as diminish tumor growth.

Another chemokine, CCL1 affects the migration of monocytes and lymphocytes and has
therefore potential to enhance autoimmunity as well as tumor immune responses. The
antagonistic aptamer developed could be used as a tool to elucidate the chemokine’s function in
disease progression and determine the therapeutic potential of this target (Marro et al., 2006).

Adhesion molecules

Adhesion molecules are an integral part of tumorigenesis in that they allow for cellular migration
through cell-to-cell contact. This can facilitate the spread of cancers. Metastasis development
occurs if tumor cells adhere to distal tissues via adhesion molecules. Such molecules include the
vascular cell adhesion molecule-1 (VCAM-1) (Chauveau et al., 2007), L-Selectin (Hicke et al.,
1996), P-Selectin (Jenison et al., 1998), as well as the lectin Sialyl Lewis X (Jeong et al., 2001).
Treatment with antagonists to these adhesion molecules could therefore inhibit the development
of metastasis. Aptamers specific to these targets have been developed. The ability to block
cellular adhesion in vitro has been demonstrated for the P-selectin (Jenison et al., 1998) and
Sialyl Lewis X aptamers (Jeong et al., 2001), whereas the function of the L-Selectin aptamer has
been validated in vivo (Hicke et al., 1996) . The aptamer was capable of blocking human PBMC
adhesion in a xenogeneic murine model. For cancer treatment, adhesion molecules are a double-
edged sword: Though adhesion molecules are involved in the spread of cancers through the
formation of metastasis, they also enhance extravasation of monocytes and macrophages into the
tumor milieu to inhibit tumor growth. This dual role is reflected in the fact that the expression of
selectins in patients has been correlated with tumor-progression, even though local tumor growth
in selectin-deficient mice is enhanced (Borsig et al., 2001; Borsig et al., 2002; Borsig, 2004).
Costimulatory receptors

Antigen stimulation is not the only component in T cell activation. T cells also express other
types of receptors, called costimulatory molecules that provide important secondary signals to
determine the outcome of antigen encounter. The importance of costimulatory signaling is
outlined in the “two-signal hypothesis”. According to the two-signal hypothesis, optimal T cell
activation requires not only the presence of antigen but also an additional signal in form of
costimulation (Sharpe and Freeman, 2002). As the name implies, this hypothesis states that two
signals are needed to activate T cell responses. Signal one involves the interaction of the T cell
receptor with a specific antigen presented via the major histocompatability complex (MHC). The
antigen dependent stimulation alone is insufficient for activation, so that the T cell becomes
unresponsive or tolerant in the absence of a costimulatory signal. However, in the presence of a
signal through the costimulatory molecules on the surface of the APC, T cells are activated.

Though this hypothesis emphasizes the importance of costimulation, it is really an

oversimplification of the T cell activation process since the strength of the primary signal
produced by antigen varies. Indeed, a “strong” (optimal) first signal through the interaction of T
cell receptor and MHC presented antigen is often sufficient to activate T cells. The secondary
signal becomes more important in the presence of “weaker” primary signals (suboptimal) and is
conveyed through receptors expressed on the surface of T cells.

In addition to positive costimulation, T lymphocytes and antigen presenting cells also interact
through coinhibitory interactions and are capable of downregulating the T cell response. Positive
signals are important for T lymphocyte activation, while negative signals lead to inhibition of
activated T lymphocytes. The presence of these two types of costimulation regulates the degree
and duration of the immune response. Most costimulatory receptors and ligands are members of
the B7/CD28 superfamily and the tumor necrosis factor receptor (TNFR) family. One such
member of the tumor necrosis factor receptor (TNFR) member is the receptor activator of NFκB
(RANK). Receptor signaling leads to osteoclast differentiation and is implicated in the
development of bone malignancies as well as enhancement of the progression of rheumatoid
arthritis (RA). An antagonistic agents therefore has the potential to treat both diseases.
Unfortunately, developed aptamers antagonist bound to other TNF receptor family members that
contained a common structural motif (Mori et al., 2004) - not the binding ligand site of the
receptor and therefore does not act as receptor antagonist.

The B7/CD28 superfamily of proteins in named for its most prominent members B71/2 and
CD28. The interaction between two receptors, CTLA-4 and CD28, and their shared ligands B7.1
and B7.2 is particularly well characterized. The ligands are expressed on dendritic cells, B cells
and monocytes. Though B7.1 and B7.2 are capable of binding both CTLA-4 and CD28, with
opposite consequences: CTLA-4 results in inhibition, whereas CD28 is a costimulatory receptor.
Our laboratory in collaboration with the laboratory of Dr. Eli Gilboa has demonstrated that an
antagonistic aptamer targeting the inhibitory receptor CTLA-4 is capable of enhancing the
efficacy of DC based tumor immunotherapy in a murine mouse model (Santulli-Marotto et al.,
2003). The potency of this aptamer antagonist was enhanced by arraying the aptamer on a
complementary DNA scaffold to increase its valency. This allows interaction with multiple
CTLA-4 receptors simultaneously. The multimerized aptamer was indeed shown to block
CTLA-4 function in vitro as well as in vivo in a tumor immunotherapy model.

CD4 is a member of the immunoglobulin superfamily and a cell surface receptor expressed on a
variety of immunological cells, most notably a subset of T cells referred to as T helper cells. This
costimulatory receptor acts to enhance the primary immune response conveyed by the T cell
receptor. In 1998, two aptamers were described that were specific to the human and rat homolog
that bind the receptor with high affinity (Kraus et al., 1998). The ligand targeting the rat receptor
was shown to inhibit CD4 function in vitro, whereas the ligand specific to the human receptor is
used as a targeting moiety to fluorescently label CD4+ cells (Davis et al., 1998) and deliver
cargo (including siRNA) to cells expressing the receptor (Guo et al., 2005).

The biological role of the transforming growth factor β receptor III (TGFβRIII) remains
somewhat poorly understood. It has been demonstrated, however, that downregulation of
receptor levels correlates with breast cancer progression. Moreover, restoration of expression of
TGFβRIII reduced tumor invasiveness and formation of metastasis. An aptamer developed to
this target actually inhibits the interaction of TGFβRIII with its ligand TGFβ2 and would most
likely not be useful for the treatment of cancer (Ohuchi et al., 2006). However, it may be useful
in elucidating the role of TGFβRIII in cancer development.

Lastly, we would like to discuss an aptamer agonist developed to the costimulatory receptor 4-
1BB (McNamara et al., 2008). This project aimed at developing a receptor agonist rather than an
antagonist. Previously, bivalent antibodies and multivalent version of natural ligands and small
molecules have been shown to induce tumor necrosis factor receptor function. Dimerization of
the generated 4-1BB aptamer by base-pairing of a single stranded extended region similarly lead
to the creation of an agonistic compound that could enhance T cell proliferation in vitro and lead
to tumor rejection when administered in vivo.

Another class of immune-stimulatory receptors has been gaining attention recently: Toll-like
receptors (TLRs) recognize various microbial and viral products, leading to activation of innate
immune responses. Some of these receptors are capable of recognizing nucleic acid ligands such
as double - (TLR 3) or single stranded RNA (TLR8) and unmethylated DNA (TLR 9) (for
review see (Takeda et al., 2003)). Aptamers to a portion of the ectodomain of TLR3 were
developed (Watanabe et al., 2006). Though the aptamers could bind to a purified construct, they
were not capable of affecting the function of HEK293 cells transfected with TLR3.

3.1.2 Aptamers in Diseases Monitoring

Viruses

Viruses are obligate intracellular parasites with a diameter of 20-300 nm. Generally, viral
particles (virions) consist of a DNA or RNA genome and a genome-protective protein coat
(capsid). Despite their simple structure, viruses can cause a variety of diseases in animals and
plants. The World Health Organization (WHO) reports that over 15 pandemic or epidemic
diseases, including the current COVID-19 pandemic, are caused by viruses. Early detection is a
crucial stage of response intervention for epidemics and allows rapid implementation of
containment measures to reduce the risk of amplification and potential international spread 54.
Relevantly, aptamer-based technology was broadly developed for the detection of virus particles
or virus-contaminated samples from humans, livestock, fish, and even plants. Indeed, more than
150 nucleic acid aptamers targeting 34 virus species, including those viruses in the WHO’s list of
pandemic or epidemic diseases have been developed (Table (Table1,1, detailed in
Supplementary material S1). Most of these aptamers were selected using viral surface proteins as
targets. However, the structures of recombinant proteins are not always identical to natural
structures. Accordingly, aptamers selected using recombinant proteins cannot bind to their
natural targets well. Other aptamers were selected using whole viral particles via nitrocellulose
membrane or by immobilizing virus particles on microbeads. Since lots of aptamers targeted
various viruses were developed, we summarized those aptamers and aptamer-based detection
examples separately, according to species of virus, in this section. One can use these aptamers to
develop detection methods directly or improve these established methods to make detection
methods more reliable and sensitive.

Influenza virus

Hemagglutinin (HA) proteins have been used for aptamer selection for influenza A and B
viruses. Using full-length glycosylated recombinant HA protein as the target, Wang and
colleagues selected a 73-mer DNA aptamer with an equilibrium dissociation constant (Kd) of
4.65 nM 71. This aptamer was further developed for influenza A virus (IAV) H5N1 detection
using QCM, SPR, or electronic aptasensor techniques 29, 31, 72-74. The detection limit can be
as low as 2-4 hemagglutination units (HAU)/50 uL, suggesting that this aptamer is reliable for
the development of a detection method for the IAV H5N1 virus. Likewise, other DNA or RNA
aptamers against HA proteins were developed, including H1N1 75-77, H3N2 78, H5N1 63, 64,
78, and IBV 79. According to the reported sequences of those aptamers against HA proteins, we
summarized the character of those sequences as “5’-GGT GNG CAR GAN RNN GTG KSN
NNN NRN NNN NNG GCA CAN SSG T-3’”.

Aptamers targeting IAV can also be selected using the whole viral particle as well. Lai and
colleagues immobilized the whole H1N1viral particle (positive selection) or IBV (negative
selection) on magnetic beads using IAV- or IBV-specific antibodies to form an integrated
SELEX microfluidic chip, resulting in a 72-mer DNA aptamer with a Kd of 55.14 nM 61. Based
on this aptamer, IAV can be detected using aptamer-trapping PCR and a fluorescent probe, with
detection limits of 0.0064 and 0.032 HAU, respectively 61, 62. Using the graphene-oxide-based
SELEX procedure, Kim and colleagues developed two H5N2 virus-specific DNA aptamers
which were then used to detect the H5N2 virus using lateral flow strips, with a limit of 1.27×105
EID50/mL in the buffer and 2.09×105 EID50/mL in the duck’s feces, respectively 80.

The above reports indicate that protein-SELEX was more popular than virus particle-SELEX for
IAV-specific aptamer selection and aptasensors were much sensitive for IAV detection. All these
selected aptamers enriched the aptamer resource for IAV detection using aptamer-based
techniques.

Human immunodeficiency virus (HIV)-1

In 2000, Yamamoto et al. developed an RNA aptamer, RNATat, targeting Tat proteins or
peptides derived from either HIV-1 or HIV-2 81. This aptamer was recently used to detect HIV-1
using an unmodified GNP-based colorimetric assay and split RNATat 82. The detection limit of
Tat protein was 10 nM. Based on RNATat, a spectrophotometric ellipsometry-based aptasensor
was developed to detect Tat protein with a detection limit of 1 pM 83. In addition, a 38-mer
DNA aptamer binding to the reverse transcriptase of HIV-1 was also developed 84, 85 and
eventually used for HIV-1 detection via radioactivity-based reverse transcriptase nucleotide
incorporation assays, with a detection limit of 100-300 virions 60.

Hepatitis virus

Using the E antigen of hepatitis B virus (HBV) as the target, DNA aptamers were developed and
applied for the detection of the E antigen with detection limits of the enzyme-linked
oligonucleotide assay and the fluorescence-based aptasensor were 0.1 ng/mL 58, 86 and 609
ng/mL 87, respectively. An aptamer targeting the surface antigen of HBV (HBsAg) was also
developed for HBV detection using a chemiluminescence aptasensor technique, with a detection
limit of 0.1 ng/mL 88. Similarly, the core antigen of the hepatitis C virus (HCV) was used as a
SELEX target to develop a 59-mer RNA aptamer and seven DNA aptamers 89, 90. The RNA
aptamer was used to detect HCV core antigen via lateral flow strip technology, with a detection
limit of 10 pg/mL 59. One of the resulting DNA aptamers was combined with electrochemical
impedance spectroscopy to detect HCV core antigen, with a detection limit of 3.3 pg/mL 91.

Bacteria

Bacteria are microscopic, single-celled organisms that exist in millions of environments, both
inside and outside other organisms. Though some bacteria are harmful, others serve useful
purposes. Harmful bacteria that cause bacterial infections and diseases are termed pathogenic.
Bacterial diseases occur when pathogenic bacteria get into the body, reproduce, and crowd out
healthy bacteria, or replicate in tissues that are normally sterile. Harmful bacteria may also emit
damaging toxins. Because of their sufficient size, bacteria can be harvested using a centrifuge
and most aptamers against bacteria can be selected with whole-cell-based SELEX.

Escherichia coli

E. coli is a type of bacteria that normally lives in the intestines of humans and other animals.
Most types of E. coli are harmless and even help to keep the digestive tract healthy, but some
strains can cause diarrhea. E. coli O157:H7 is a very threatening strain that causes abdominal
cramps, vomiting, bloody diarrhea, and even acute kidney failure in children. Several DNA
aptamers targeting E. coli have been developed using lipopolysaccharide (LPS) or whole-cell
O157:H7 as the target. Using the US patent-originated aptamers 144, Wu et al. established an
aptamer-based colorimetric detection method for O157:H7 using truncated DNA aptamers
against LPS, with a detection limit of 10,000 CFU/mL 145. Based on these aptamers,
electrochemical aptasensor and lateral flow strip assay for O157:H7 detection were
invented 146, 147, lowering the detection limit to 10 CFU/mL. These reports indicate that these
aptamers published in the US patent can be further applied for the development of commercial
products. Another 45-mer DNA aptamer targeting whole-cell O157:H7 was developed using
cell-SELEX and used for detection via QCM-sensor technology, with a detection limit of 1460
CFU/mL 124. A 72-mer DNA aptamer was immobilized on magnetic beads to capture O157:H7
in blood samples for detection 148. The same DNA aptamer was used in a hydrothermally grown
ZnO nanowire array to construct a high-performance photoelectrochemical aptasensor to detect
149
O157:H7, with a detection limit of 1.125 CFU/mL . Comparing to clinic detection methods,
these aptamer-based detection techniques reduced the detection time and increased the detection
sensitivity.

In 2008, Bruno et al. developed DNA aptamers against LPS from E. coli O111:B4 using SELEX
against LPS-conjugated magnetic beads 150. Subsequently, some of these aptamers were used to
detect E. coli LPS. Xie et al. developed an aptasensor based on a hybridization chain reaction for
LPS detection with a detection limit of 1.73 ng/mL 151. Zhu et al. employed a dual aptamer-
152
functionalized GNP probe to detect LPS, reaching a detection limit of 1 μg/mL . DNA
aptamers targeting other features of E. coli were developed and broadly applied for E.
coli detection. Bruno et al. described a high-throughput DNA aptamer screening and detection
method based on competitive FRET technology 123. With this method, they obtained several
DNA aptamers targeting the outer membrane proteins of E. coli 8739 and established an E.
coli 8739 detection method with a detection limit of 30 CFU/mL in binding buffer solution.
DNA aptamers against the fimbriae protein of enterotoxigenic E. coli K88 were developed with a
Kd of 25 nM and may be further applied for E. coli K88 detection 153.

Using whole cells of E. coli as the SELEX target, Kim et al. isolated 28 DNA aptamers that bind
to whole-cell E. coli. Among these, four were further characterized with K d values ranging from
12.4 to 25.2 nM 154. These aptamers were combined as a cocktail to detect E. coli with a
detection limit of 370 CFU/mL 155. Using the same aptamers, Jin et al. developed a detection
156
method based on FRET aptasensor technology and reached a detection limit of 3 CFU/mL .
Likewise, Hua et al used the same aptamers to construct a sensitive potentiometric resolved
ratiometric photoelectrochemical aptasensor for E. coli detection with a detection limit of 2.9
CFU/mL 157.

Salmonella

Salmonella is the type of bacteria most frequently reported as causing food-related illness in the
United States. People and animals can carry salmonella in their intestines and feces. The bacteria
often spread through contaminated foods. As such, rapid and sensitive detection is of critical
importance. Joshi et al. selected a DNA aptamer targeting the outer membrane proteins of S.
enterica serotype Typhimurium and obtained five aptamer candidates. Using one of the aptamers
(5'-TAT GGC GGC GTC ACC CGA CGG GGA CTT GAC ATT ATG ACA G-3'), they
developed an aptamer-based trapping PCR detection method with a detection limit of 1 CFU/mL
in rinsate samples 46. This aptamer was broadly applied in various detection methods based on
different aptasensor technologies, with detection limits ranging from 1 to 1000 CFU/mL 47, 158-168,
suggesting that this aptamer is worth to be applied to develop commercial products. In addition,
using Vi-antigen as the target, Pathania et al. developed a DNA aptamer using a microtiter-based
SELEX approach. This aptamer was applied as an electrochemical aptasensor for Vi-antigen
detection with a limit of 100 pg/mL 134.

Since bacteria can be collected by centrifuge, many aptamers have been developed using cell-
SELEX. For example, Duan et al. developed a DNA aptamer using cell-SELEX and immobilized
this aptamer on magnetic beads for S. Typhimurium capture. Then, the bead-captured S.
Typhimurium could be detected by the FAM-labeled aptamer using a fluorescence
spectrophotometer. The detection limit of this aptamer-based fluorescent assay for S.
typhimurium was 25 CFU/mL 169. This design is quite simple and even can be used to detect S.
typhimurium by using a magnet and a fluorescent excitation lamp. However, due to the same
aptamer was both used to capture and fluorescent development, there is a binding competition
between the immobilized aptamer and the fluorophore-labeled aptamer, resulting in low
sensitivity in detection. Therefore, two different aptamers used in this design should improve the
detection sensitivity and specificity. Besides, if the detection aptamer can be labeled with other
dyes that can be observed by the naked eye, the detection assay should be more convenient.
Another example, Wang et al. obtained a 90-mer DNA aptamer using QCM-based SELEX and
developed a QCM-based aptasensor for S. Typhimurium detection with a limit of 1000
CFU/mL 170. To improve the detection sensitivity, they further applied this aptamer for S.
Typhimurium detection using aptamer-based PCR and electrochemical impedance aptasensor
technologies 135, 171, lowering the detection limit to 100 CFM/mL and 80 CFU/mL, respectively.
A series of high-binding affinity DNA aptamers against the whole bacterial cells of S.
enteritidis and S. typhimurium were developed 172, 173. Later, those aptamers were used in a
fluorometric GO-based assay for S. enteritidis detection with a limit of 25 CFU/mL 39 and a
fluorescence-based assay for S. typhimurium detection with a limit of 20 CFU/mL 173.

Mycobacterium tuberculosis

M. tuberculosis is the causative agent of tuberculosis, one of the top 10 causes of death globally.
Aptamers targeted to different proteins of M. tuberculosis were developed. Qin et al. developed
DNA aptamers against the MPT64 antigen 174. These aptamers were then applied for an
electrochemical aptasensor for ultrasensitive detection of M. tuberculosis with a limit of 20
fg/mL in human serum samples 175. Ansari et al. identified an 80-mer aptamer that binds to the
FbpA protein of M. tuberculosis with high affinity using protein-SELEX 127. They further applied
this aptamer in a GO-based fluorometric assay for FbpA detection, with a limit of 2.1 nM in
human serum samples. Dhiman et al. developed a 28-mer DNA aptamer against the biomarker
of M. tuberculosis, HspX antigen, using protein-SELEX 128. This aptamer was used for M.
tuberculosis infection diagnosis via ELONA and electrochemical sensor systems, with a
sensitivity as high as ELISA and qPCR, but with a lower cost 129, 130, 176.

Fungi

There are millions of fungal species, but only a few hundred can cause human illness. Fungal
infections are common throughout much of the natural world. In humans, fungal infections occur
when an invading fungus takes over an area of the body and overwhelms the immune system.
Compared to viral and bacterial infections, fungal infection is uncommon and unlikely to be life-
threatening. The CDC divides fungal diseases into four types: (1) common fungal diseases,
including fungal nail infection, vaginal candidiasis, ringworm, and Candida infections of the
mouth, throat, and esophagus; (2) fungal diseases that affect people who live in or travel to
certain areas, including blastomycosis, coccidioidomycosis or valley fever, Cryptococcus
gattii infection, histoplasmosis, and paracoccidioidomycosis; (3) fungal diseases that affect
people with weakened immune systems, including aspergillosis, Cryptococcus
neoformans infection, Candida auris infection, mucormycosis, Pneumocystis pneumonia,
and talaromycosis; (4) other fungal diseases, including fungal eye infection, mycetoma, and
sporotrichosis.

To detect fungal diseases, aptamers have largely been developed against mycotoxins, which are
toxic secondary metabolites produced by fungi. Based on these aptamers, many promising
aptasensors have been developed for the detection of various mycotoxins 213-215. Detection of
mycotoxin contaminants, and pathogenic fungi are important for public health. Nucleotide
aptamers targeting infectious fungi have also been developed. Tang et al. developed two high-
affinity DNA aptamers specifically targeting (1→3)-β-D-glucans from the cell wall of Candida
albicans using SELEX 216. These two aptamers were used to establish an ELONA assay for
(1→3)-β-D-glucans detection in serum samples.

Parasites

Parasites are organisms that live off other organisms (hosts) to survive. Some parasites can make
their hosts sick, resulting in parasitic diseases 217. Parasitic infections are common in tropical and
subtropical regions of the world. There are three main classes of parasites that cause disease in
humans: protozoa, helminths, and ectoparasites.

Plasmodium, a kind of protozoa and the causative pathogen of malaria, causes more deaths
globally than all other parasitic diseases. Several aptamers targeting Plasmodium have been
developed and some have been used for diagnosis. Most Plasmodium-specific aptamers have
been summarized previously 218, excluding a few novel aptamers with malaria diagnosis
applications. To select an aptamer targeting P. falciparum, Singh et al. immobilized P.
falciparum glutamate dehydrogenase (PfGDH, for SELEX) or human glutamate dehydrogenase
(hGDH, for counter-SELEX) onto PVDF membranes for protein-SELEX 219. They obtained a 90-
mer DNA aptamer with a binding affinity of 0.5 μM. To detect PfGDH in serum samples, they 1)
219
coupled this aptamer with carbon dots as the probe , 2) coupled the aptamer with dye as the
catcher for an enzyme-catalyzed reaction 220, and 3) applied this aptamer as the capture in a field-
effect transistor biosensor 221, resulting in detection limits of 2.85 nM, 63.97 pM, and 48.6 pM,
respectively. For rapid diagnostic tests for Plasmodium infection, Joseph et al. identified high
mobility group box 1 protein (HMGB1) as a biomarker for Plasmodium infection using gene
222
expression databases, ribosome profiling analysis, and structural modeling . Later, they
immobilized biotinylated HMG-box onto streptavidin-coated magnetic beads for protein-SELEX
and developed several DNA aptamers of potential use for malaria diagnosis. Minopoli et al.
developed an antibody-aptamer plasmonic biosensor to detect P. falciparum lactate
dehydrogenase (PfLDH) by immobilizing PfLDH-specific antibodies onto gold nanoparticles for
223
PfLDH capture and labeling the PfLDH-specific aptamer to detect the captured PfLDH . Using
this aptasensor, the detection limit was as low as 1 pg/mL in blood samples. DNA aptamers
targeting the surface of erythrocytes infected with P. falciparum were developed using cell-
SELEX as well 224, 225.

Trichomonas vaginalis, an anaerobic, flagellated protozoan parasite, is the causative agent of

trichomoniasis. Espiritu and colleagues selected a DNA aptamer against the T.
vaginalis adhesion protein AP65 using SELEX and developed an enzyme-linked aptamer assay
to detect T. vaginalis with a detection limit of 8300 cells/mL 226.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis. Aptamers

against L. infantum were developed by different groups and summarized 218. Frezza et al. isolated
two DNA aptamers previously identified in an ssDNA library against L. infantum H3 (LiH3)
protein. They labeled either of the two aptamers with digoxigenin for LiH3 binding and used an
HRP-labeled anti-digoxigenin antibody for detection. Their results indicated that these two DNA
aptamers are promising biosensing molecules for leishmaniasis diagnosis 227.

Cryptosporidium is a microscopic parasite that causes the diarrheal disease cryptosporidiosis. To

detect Cryptosporidium parvum oocysts in fresh foods, Iqbal et al. developed 14 DNA aptamers
with high affinities for C. parvum oocysts and created an electrochemical aptasensor to detect C.
parvum oocysts in fresh fruit and, raw lake and river waters samples, with detection limits of 100
and 50 oocysts, respectively 228, 229.

Toxoplasma gondii is a protozoan parasite that infects most species of warm-blooded animals,
including humans, and causes the disease toxoplasmosis. Diagnosis of toxoplasmosis is typically
made by serologic testing, namely immunoglobulin G (IgG) detection. Luo et al. obtained two
DNA aptamers targeting anti-toxoplasma IgG by SELEX and developed a QD-based aptasensor
230
for anti-toxoplasma IgG detection in serum samples, with a detection limit of 0.1 IU . Vargas-
Montes et al. developed two DNA aptamers against T. gondii rhoptry protein 18, a major
virulence factor, from the N40 ssDNA library using protein-SELEX. Based on these two
aptamers, they established an ELONA assay for ROP18 protein detection in serum samples, with
a limit of 1.56 μg/mL 231. Shen et al. used protein-SELEX to develop DNA aptamers against
the T. gondii surface antigen protein, a key marker for laboratory diagnosis, from the ssDNA
microsphere library modified with indole-dU (w), phenol-dU (Y), and amine-dU(X).
Furthermore, they used one of the identified aptamers as the probe in an ELONA assay to detect
the native surface antigen protein secreted by T. gondii in mouse and human serum samples, with
a detection limit of 1.56 μg/mL 232.

3.2 Aptamers and the Diseases they treat

DNA aptamers have been selected predominantly with cell-SELEX and magnetic beads-based
methods by targeting various viruses and bacteria or their antigen protein [6], including targets
such as norovirus, influenza virus, severe acute respiratory syndrome coronavirus (SARS-CoV),
hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human
papillomavirus (HPV), salmonella typhimurium, and pathogenic E. coli. Because of the low cost
and short production time of DNA aptamers, the DNA aptamer based diagnostic tools hold
particular advantages for the diagnosis of infections that need point-of-care testing, such as
infections caused by highly pathogenic pandemic influenza virus, HIV, SARS-CoV and malaria.
Avian influenza virus H5N1 is a highly pathogenic subtype of the influenza virus, which can also
infect humans and has a high mortality rate. Wang et al. selected a DNA aptamer targeting H5N1
using both the virus antigen and the whole virus particle using the SELEX method [67]. For the
first four selection cycles, the purified virus antigen hemagglutinin (HA) was used as the target
protein, while for the remaining eight cycles the entire H5N1 virus particles were used as the
targets. Using such a mixed selection mode might be a good strategy to obtain ideal aptamers,
which can overcome the drawbacks of selection using only free biomarkers or cells.
In the case of cardiovascular diseases, markers in the blood, such as myoglobin, C-reactive
protein, L-homocysteine and thrombin have been used to select DNA aptamer for the diagnosis
of related diseases. Myoglobin increases after acute myocardial infarction, which is an important
early marker in urgent diagnosis of cardiovascular diseases. Wang et al. selected DNA aptamers
against myoglobin using a fluidic chip method [72]. The DNA aptamer with the lowest Kd value
(4.93 nM) was subjected to the development of different biosensors for the detection of
myoglobin, including the Myoglobin-induced structural switching supersandwich biosensor [72]
and the antibody-Myoglobin–aptamer sandwich biosensor [76]. Human C-reactive protein (CRP)
is a homopentameric oligoprotein, which has been validated as a powerful predictor and risk
factor of inflammation and cardiovascular disease [77]. Yang et al. selected a DNA aptamer
(Kd = 3.9 nM) targeting CRP and used it to develop a sensor based on surface plasmon resonance
technology [78]. L-Homocysteine is an amino acid intermediate, whose elevated level in the
blood is associated with coronary heart disease [79]. McKeague et al. selected DNA aptamers
targeting L-homocysteine and developed an aptamer-AuNP sensor, in which the DNA aptamers
first coil around the surface of the gold particles and release the particles after binding to the
homocysteine, which leads to salt-induced aggregation and colorization of the particles [73].
Thrombin is a serine protease that plays a critical role in the formation of obstructive blood clots,
or thrombosis, which is involved in various diseases including chronic cardiovascular diseases
[80]. The first reported DNA aptamer is the thrombin aptamer. Since then, thrombin aptamers
have been used as model aptamers to explore the mode of aptamer-based biosensors. It is the
most frequently used DNA aptamer for the demonstration of the proof-of-principle of various
detection methods [81].

3.3 GINATOP (AS1411)

AS1411, formerly known as AGRO100, is a quadruplex-forming oligonucleotide aptamer

application that binds to nucleoli. AS1411 (core sequence: 5′-
GGTGGTGGTGGTTGTGGTGGTGGTGG-3′) is rich in the DNA sequence of G, physiological
conditions can form G4 structure [46, 60]. AS1411 can cause toxicity when entering cancer cells,
so it can directly kill cancer cells without adding any killing agent [42, 43, 61,62,63,64]. AS1411
may be one of the most promising nucleic acid sequences in G-quadruplex. AS1411 was
incidentally found to have good nuclease resistance and thermal stability. In addition, it has
shown certain anti-proliferative activity in almost all cancer cell lines, so it seems to have a wide
range of therapeutic potential, and it is also the first anticancer adaptor and the first known drug
specifically targeting nucleoprotein, and the phase I clinical study on it has also achieved good
results [46]. Although AS1411 has a good targeted therapeutic effect, the mechanism of its
cancer targeting action is not fully understood [43, 46, 65]. Other forms of DNA can exist
alongside the classical double helix as viable molecular targets. In the past decade, the telomere
G-quadruplet structure has been considered as a potential target for the discovery and
development of novel anticancer therapies. AS1411 is a landmark G-quadruplet structure in the
study of g-quadruplet structure, folding and polymorphism. Significant progress has been made
in the mechanism of selective interactions between double-stranded DNA and telomere G-
tetrachromes [65].

3.3.1 Ginatop Mechanism In Cancer Treatment

Numerous research groups have used AS1411 as a targeting agent to deliver nanoparticles,
oligonucleotides, and small molecules into cancer cells. Studies in animal models have
demonstrated that AS1411-linked materials can accumulate selectively in tumors following
systemic administration. The mechanism underlying the cancer-targeting ability of AS1411 is
not completely understood, but recent studies suggest a model that involves:

(1) initial uptake by macropinocytosis, a form of endocytosis prevalent in cancer cells;

(2) stimulation of macropinocytosis by a nucleolin-dependent mechanism resulting in further

uptake; and
(3) disruption of nucleolin-mediated trafficking and efflux leading to cargoes becoming trapped
inside cancer cells.

3.3.2 Targeting Mechanism Of Ginatop In Cancer Treatment

Passive targeting

Passive targeting is mainly to modify the aptamer to the surface of the nanocarrier which can
carry drugs to form the aptamer targeting nanocarrier. The growth and metabolism of tumor
tissue is extremely vigorous, and vascular remodeling is incomplete. There is a gap between
vascular endothelium of 10–1000 nm, so only particles within this range can reach the tumor site
through blood circulation, through EPR effect, namely the high permeability and retention effect
of solid tumor. Accumulate in tumor site is therefore whether can realize the EPR effect
researchers have designed passive targeting carrier directly measure, the particle size of nano
carrier starts by the kidneys to filter out, too easy to be liver intake rather than in the tumor
position, therefore the particle size of nano carrier must be within the scope of a specific
clearance can penetrate vascular endothelial cells, through passive targeting to enter tumor tissue,
The structure and status of micro vessels in tumor sites also have a great influence on EPR effect
[215, 216]. A novel aptamer-modified immunolipoprotein nanostructure for drug delivery and
scale-up chemotherapy immunotherapy was proposed in 2018 [217]. The lipophilic aptam-
immune adjuvant CpG fusion sequence (Apt-CpG-DSPE) was combined to facilitate the
modification of high-density lipoprotein (HDL), and then doxorubinin (Dox) was sequentially
embedded into the consecutive base pairs of Apt-CpG. imHDL/Apt-CpG-Dox was synthesized.
This immune HDL nanotaph causes a degree of site-specific structural collapse in the material
between the tumor cells that inspire HDL biological function. At this point, under the recognition
condition of AS1411 and nucleolus, the dissociated Apt-CpG-Dox would be endocytosis into
tumor cells and transfer Dox to the nucleus, and the released CpG motifs would further trigger
antigen recognition, resulting in tumor ablation and antigen release as much as possible, while
inducing the secretion of a large number of pro-inflammatory cytokines. So as to enhance the
host's anti-tumor immunity.

Active targeting

Active targeting is mainly to directly connect the aptamer with the drug/fluorescent substance to
form the conjugate of aptamer-drug/fluorescent substance. Pass drug delivery system is mainly
based on the receptor and the ligand specificity combined into tumor cells, most species of tumor
cells contain high compared to normal cells express or express receptors, so the corresponding
ligand modification on the surface of nanometer carrier, can increase the tumor cells on the
specificity of the vehicle recognition, greater intake of carrier, The targeted drug release can be
carried out more accurately at the tumor site, with minimal or no toxicity to non-tumor tissues. In
addition to improving drug availability, tumor treatment can be more effective. At present, active
targeting drug delivery system is the most common to target tumor cells
[16, 77, 218,219,220,221]. Mona Alibolandi group [84] developed a molecular drug delivery
system for the targeted treatment of non-small cell lung cancer (NSCLC). This system uses
gemecitabine (GEM) loaded AS1411 apper surface modified polyethylene glycolic acid (lactic
acid co-glycolic acid) as nano polymer (Apt-GEM-NP).

3.4 Challenges In Aptamers application

1. Aptamer degradation

The rapid degradation of aptamers (especially RN A aptamers) by nucleases in biological media,

and in blood in particular, is a serious problem that puts limits on their practical application. The
average time of oligonucleotide decay in blood ranges from several minutes to several tens of
minutes depending on the oligonucleotide concentration and conformational structure. Since
such a short time range is unacceptable for most therapeutic applications, several methods for
protecting aptamers against degradation by nucleases have been developed.

One of the conventional methods used to generate nuclease-resistant aptamers is by performing

SELEX with oligonucleotides containing modified nucleotides (Fig. 2). Special DNA and RN A
polymerases that are able to utilise nucleoside triphosphate substrates with a modified, for
example, 2’ sugar position are used to generate such oligonucleotides. 2’-Amino pyrimidine
nucleosides [20, 21], 2’-fluoropyrimidine nucleosides [22, 23], 2’-O-methyl purine, and 2’-O-
methyl pyrimidine nucleosides [24, 25] are currently used for this purpose. The only aptamer
approved for medical application known as Macugen (Fig. 3) is a vivid example of an
oligonucleotide modified using this approach [26]. Modification of nucleotides already included
into aptamers could also be performed after the SELEX procedure; however, the inclusion of
additional functional groups in this case can affect the specificity and affinity of an aptamer.
Nevertheless, some modifications can increase aptamer resistance to nucleases without affecting
their binding to target molecules. The most common and effective type of such aptamer
improvements is the modification of 3’- and 5’-nucleotides [27]. Sometimes unmodified
aptamers demonstrate very high resistance to degradation by blood nucleases [28]. This feature
might be provided by the formation of specific three-dimensional structures that protect the 3’-
and 5’-termini of aptamers against exonucleases.
Fig. 2

Most frequently used modifications of nucleotides providing resistance of aptamers to nuclease

degradation
Fig. 3

The structure of the first FDA-approved aptamer, Macugen. The following modified nucleotides
were used: f – 2’-fluoronucleotide, m – 2’-O-methylnucleotide. The aptamer was conjugated to
40 kDa PEG to avoid quick excretion during renal filtration.

2. Aptamer excretion from the bloodstream by renal filtration

The removal of aptamers from the bloodstream via renal filtration complicates their therapeutic
application. Most aptamers have a molecular weight ranging from 5 to 15 kDa (15-50
nucleotides), and they can be easily excreted by kidneys capable of removing substances with a
molecular weight below 30-50 kDa. Conjugation of aptamers with polyethylene glycol (PEG)
with a molecular weight of 20 or 40 kDa is the most common solution to this problem ( Fig. 3).
This method is currently being used to increase the bloodstream circulation time not only of
oligonucleotides, but also of proteins, peptides and low-molecular-weight substances [36, 37].
The PEG-conjugated aptamers are excreted from the bloodstream slowly (up to several days) and
do not lose their specificity. And, besides, such PEG-conjugated aptamers are more effectively
delivered to tissues and organs [38, 39]. As an alternative, aptamers could also be conjugated
with cholesterol molecules. This modification also prolongs aptamer circulation in the
bloodstream [40].

3. Control of the duration of action

The pharmacokinetic parameters of a drug (e.g., action duration) are very important in its
therapeutic application. The duration of action depends on multiple factors, including
degradation, involvement in metabolic processes, renal excretion, etc. All these factors should be
taken into consideration before drug prescription, and sometimes they limit its application. The
use of aptamers as drugs can often solve the problems associated with controlling the duration of
action. One of the possibilities is to generate antidotes to aptamers by synthesizing a
complementary oligonucleotide. Hybridization with antidote causes a change in aptamer
conformation and complete loss of its ability to bind the target molecule (fig.4). The efficiency
of this approach has been confirmed by experiments on animal models. An aptamer was
delivered into the bloodstream and exhibited a therapeutic effect, while subsequent injection of
an antidote inactivated the aptamer and stopped its action [41, 42]. The high efficiency of
aptamer hybridization with an antidote in blood provides a unique opportunity to control the
duration of the therapeutic action. It makes the application of aptamers preferable, since it is
either impossible or very difficult to control the duration of action of antibodies or low-
molecular-weight substances.
Fig. 4

Antidote-dependent regulation of aptamer functioning. The aptamer 9.3t is shown as an example

[77]. This aptamer interacts with the coagulation factor IXa and has anticoagulation properties.
Administration of a complementary antidote leads to quick inactivation of this aptamer and
restoration of blood coagulation

Another effective and inexpensive way to control aptamer activity in the bloodstream without the
necessity to generate a unique antidote is through the application of polycationic biopolymers
that effectively bind polyanionic oligonucleotide molecules [43, 44]. Numerous polymers
originally developed for gene therapy and delivery of DNA or siRN A possess the ability to bind
NAs [45, 46]. Some low-molecular-weight molecules, such as porphyrin, can also bind particular
conformational structures and inactivate an aptamer [47]. The blood does not contain significant
amounts of NA due to the high activity of nucleases; therefore, it is presumed that biopolymers
will bind preferentially foreign NAs (in particular, aptamers).

4. Interaction of aptamers with intracellular targets

Most aptamers were selected using molecules located on the cell surface or in the bloodstream.
This potentially makes their application rather easy, since all that is needed to trigger the
therapeutic effect is to deliver the aptamer into the bloodstream. However, some advances in the
intracellular delivery of aptamers have recently been achieved. Special expression systems are
able to generate aptamers inside cells and ensure their accumulation either in nucleus or in the
cytoplasm. For example, transfection of cells with a recombinant vector expressing the aptamer
sequence under a U6 promoter allows specific inactivation of nuclear target proteins [50, 51],
while aptamer expression under a tRN A promoter ensures predominantly cytoplasmic
localization of aptamers [52]. Cell-type-specific aptamer synthesis can be achieved by using
directional viral expression systems that deliver vectors to particular cells [53, 54]. The
concentration of expressed aptamers (also known as intramers) can be increased not only by
using strong promoters that ensure a high level of expression, but also by limiting the rate of
aptamer degradation by nucleases through protection of the 3’- and 5’- termini with additional
structures (e.g., hairpins) [50].

Another way of delivering aptamers to intracellular target molecules is by the transfer of

aptamers from the bloodstream to cells through receptor-dependent endocytosis [55, 56]. For
example, endocytosis of aptamer binding prostate-specific membrane antigen (PSMA) provides
effective and specific delivery of conjugated drugs to cancer cells expressing this antigen on their
surface [57, 58].

5. Generation of aptamers using unpurified target proteins

Aptamer generation in most cases requires the availability of purified target molecules. Protein
target molecules are expressed in cell cultures and purified by affine chromatography. These
procedures are timeand labor-consuming, thus delaying the production of corresponding
aptamers. Moreover, some proteins are difficult to purify due to their chemical properties.
Sometimes aptamers generated against target proteins expressed in prokaryotic cells do not
interact with the same proteins expressed in eukaryotic cells due to post-translational
modifications. These modifications can make epitopes of eukaryotic proteins inaccessible to
aptamers generated against the proteins expressed in prokaryotic cells [59].

The modified SELEX protocol (Cell-SELEX) can be used to select aptamers that recognize cell-
surface proteins [60, 61] (fig.5). Cell-SELEX allows to select aptamers located directly on the
surface of live cells. It is also possible to generate aptamers that recognize specific
microorganisms (e.g., such parasites as trypanosomes) [62, 63]. Cell-SELEX includes a negative
selection step with a cognate cell type or cell line negative for target markers. One of the
advantages of Cell-SELEX is that it does not require exhaustive information about cell-specific
protein markers. The combination of negative selection with normal cells and positive selection
with transformed cells will provide aptamers specific to tumor markers and promote the
development of early cancer diagnostics.
 CHAPTER FOUR

CONCLUSIONS
Aptamers continue to grow as viable clinical agents. They represent a unique class of molecules
that are larger than small molecule drugs, but smaller than antibodies. Importantly, they have
unique pharmacokinetic properties which could be exploited for various purposes. One highly
attractive feature of aptamers is they appear to be non-immunogenic. Moreover, they are
amendable to chemical modification and bioconjugation to various moieties such as
nanoparticles, imaging agents, siRNAs and therapeutic drugs. As discussed above, they are
proven clinically as inhibitors with great promise as targeting ligands for treating or diagnosing
cancer, HIV, and other diseases.

As with any pharmacologic agent, aptamer translation faces some obstacles. One initial decision
is which nucleic acid sugar is best suited for the specific application. Both DNA and RNA
aptamers are capable of binding a target with high specificity and selectivity. RNA aptamers are
able to form more stable 3D structures due to the strong intrastrand RNA-RNA interactions, yet
DNA aptamers have better chemical and thermodynamic stability due to its B-form helix
structure. Therefore, RNA aptamer pools may yield more structurally distinct agents resulting in
a quicker selection. However, this may comes at a cost since RNA aptamers require modified
oligonucleotides. The cost to produce aptamers containing the commonly used modified
oligonucleotides for stabilization is rather expensive, although the cost is decreasing. For
example, the cost of 2′-fluoro, 2′-ribo and 2′-O-methyl RNA phosphoramidites are
approximately $20 per gram[85]. Increased demand or new synthesis technology will likely
further reduce these costs. Another current barrier is the difficulty to synthesize longer modified
aptamers. Current oligonucleotide synthesis technology is limited to 50-60 nt making the larger
aptamer siRNA chimeras difficult to translate into clinical practice as chemically produced
agents. Advances in nucleotide synthesis technology will likely also overcome this limitation.

Biomarkers play a critical role in improving the drug development process as well as in the
larger biomedical research enterprise. Understanding the relationship between measurable
biological processes and clinical outcomes is vital to expanding our arsenal of treatments for all
diseases, and for deepening our understanding of normal, healthy physiology. Since at least the
1980s, the necessity of using biomarkers as surrogate outcomes in large trials of major diseases,
such as cancer [10] and heart disease [11], has been widely discussed. The FDA continues to
promote the use of biomarkers in basic and clinical research, as well as research on potential new
biomarkers to use as surrogates in future trials [12]. However, for all their potential to do good—
to speed drug development, to reduce exposure to ineffective experimental treatments, and so on
—biomarkers present substantial risks when trial designers confuse them with clinical endpoints.

Biomarkers could only serve as true replacements for clinical relevant endpoints if we
completely understood the normal physiology of a biological process, the pathophysiology of
that process in the disease state, and effects of an intervention – pharmacological, device, or
otherwise – on these processes. Since we rarely if ever have the full picture of those types of
processes, since there are always more details we don’t know or understand, biomarkers as
surrogate endpoints need constant reevaluation. Studies using biomarkers should always have as
ultimate measures clinical outcomes, at least for retrospective analysis of biomarker correlation
success. Without continual reevaluation of the relationship between surrogate endpoints and true
clinical endpoints, we risk again approving whole classes of drugs that either have no additional
benefit or, worse, that harm patients.

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