SCI ED 106

Biochemical and Biophysical

Source: www.elsevier.com/locate/ica

Submitted to: Prof. Nicetas Ruiz

Submitted by: Jemimah Joy I. Guarin

It emphasizes the research on metal-binding properties of catalytic DNAzymes, to

understand the structural properties leading to metal ion specificity. DNAzymes, also known as

deoxyribozymes, are oligomers of DNA whose sequence provides them with catalytic

functionality. The specific type of catalytic functionality for certain DNAzymes depends on their

DNA sequence. Still, in most cases, they function as cofactors, such as metal ions, the most

common cofactors for DNAzyme catalysis. In addition, DNAzymes are a class of metalloenzymes

that perform many different reactions with rates that can compete with other known types of

enzymes. DNAzymes are known to bind metal ions due to their highly negatively charged

phosphodiester backbone. It has also been identified that DNAzymes bind to the metal ion

selectively, resulting in the conversion of DNAzymes to sensors capable of reporting on the

presence of the necessary metal ion cofactor in environmental samples as within cells.

This research also involves the biochemical approaches toward understanding metal ion

selectivity for the divalent metal ion-dependent DNAzymes and the trivalent and monovalent

metal ion-dependent DNAzymes. For the divalent metal ion, the first DNAzyme isolated in vitro

was the Pb2+ - dependent DNAzyme, which was named GR5 DNAzyme, and an in vitro selection

of DNAzymes was also carried out in which DNAzymes have the capability to cleavage in all

RNA substrates in the presence of Mg2+, resulted to two structurally distinct scaffolds named 8-17

and 10-23 DNAzymes. Of the two, 8-17 display much higher activity in the presence of Pb2+,

which was utilized to create the first DNAzyme-based catalytic beacon sensor for lead ions. On

the other hand, the selectivity of GR5 for Pb2+ over other metal ions is considerably more

significant than the 8-17 DNAzyme. The primary barrier considered in means of the practical

application of DNAzymes for the detection of metal ions is the hybridization and dehybridization
of the DNA binding arms because they are highly temperature dependent, and to overcome this

mutagenesis of the 8-17 DNAzyme was made which resulted to a more temperature resistant

DNAzymes. On the other hand, trivalent lanthanide ion such as Ce3+, Eu3+, and Yb3+ was used to

identify several sequences for the metal ions’ activity. It was determined that two sequences were

found to be active even in the absence of Zn2+. It had a significant selectivity for Yb3+ as compared

to other lanthanide ions. Ce13 DNAzyme was one of the sequences found to have a vast substrate

scope, showing high activity with trivalent lanthanides.

Also, A43 DNAzyme, a sequence responding to a monovalent metal ion, catalyzed RNA

cleavage, specifically in the presence of Na+. This DNAzyme was truncated and converted into a

catalytic sensor to measure intracellular sodium concentrations in HeLa cells. Biochemical studies

using modified nucleotides were also conducted; these modified or unnatural bases allowed to tune

of individual functional groups of nucleotides with exceptional precision. Hence, it can be used to

explain the role of specific functional groups in enabling catalytic activity and in interacting with

metal ion cofactors. Information about kinetic analysis of metal specificity can be used to

investigate the substrate scope of a given DNAzymes and the specificity of the DNAzyme towards

a particular metal cofactor, as well as a hint to its mode of action. CE13 DNAzyme, for instance,

has a linear rate dependence on both Ce3+ and Na+, which suggests that the two metal-binding sites

are independent of each other.

The interaction of hemin-dependent peroxidase DNAzymes is fast and reliable compared

to natural peroxidases and P450 monooxygenases, mainly due to the effect of mutagenesis or

changes in the enzyme structure. With regards to the reaction product analysis, mass spectroscopy

is a very applicable method for it can identify the reaction products of a DNAzyme and also can

provide an understanding of how metal ion affects or controls DNAzyme catalysis. NMR, x-ray
crystallography, photocrosslinking, and photocleavage also did structural characterization of

DNAzymes. However, the structural determination of DNAzymes is underexplored, and reports

of NMR structures of DNAzymes are rare. But, the x-ray crystal structure of 10-23 DNAzyme

revealed an interesting nucleic acid fold in which the helices formed two four-helix junctions

stabilized by extensive base-stacking interactions and possibly by metal ions. But it was also found

that this structure was not an active form of DNAzyme. Thus, the structural explanation of the 10-

23 DNAzyme mechanism of the role of metal ions was limited.

On the other hand, the photocrosslinking method was used by several studies to obtain

information on the global fold of DNAzymes, the required features for DNA-metal interaction,

and subsequent activity. However, it may not be as direct as crystal or NMR structure, but it sheds

light on several structural aspects of metal-mediated DNA activity. In addition to the structural

characterization of DNAzymes, many spectroscopic techniques have begun to characterize the

metal-binding site of the DNAzymes, such as x-ray absorption spectroscopy (XAS), fluorescent

resonance energy transfer (FRET), circular dichroism (CD), and sensitized luminescence. These

methods are mainly metal-ion-focused techniques adapted from inorganic and bioinorganic

chemistry and are widely used to explain inorganic complexes, metalloproteins, and Metallo-

ribozymes, which can provide information on the metal-binding sites in DNAzymes.

The authors point out that the scientific field already has a rich knowledge of the interaction

of metals with proteins and RNA; however, DNA-metal interactions are far less understood.

Therefore, the authors recommended that studying more about the DNA-metal interactions is

essential in improving our understanding of the class of metalloenzymes. The methods and

techniques for characterizing metal ion-DNAzyme interactions have been applied to understand

the class of metalloenzymes betters. The authors also pointed out that there has been an increased

usage of DNAzymes as metal ion sensors both for environmental samples and, recently, for

cellular detection, ion which showed an increased interest in the properties underlying their

function and selectivity. The authors believed that achieving a better understanding of the metal

ion selectivity in DNAzymes not only opens new avenues in the field of bioinorganic chemistry,

hence, will allow to design and to improve the metal ion sensors.

The authors recommended the following future directions to achieve the full potential of

metallo-DNAzymes because the field will vastly benefit from it follows:

1. Comparative analysis of DNAzymes with similar metal ion specificity. The authors

emphasized that using the next-generation sequencing technology may make it possible

to track large-scale changes in the pool of sequences in the in vitro selection process.

Also, this method would give more information about the gain or loss of particular

domains as a property of the selection round and for comparative analysis.

2. Incorporation of modified nucleotides to study structural features responsible for metal

selectivity and activity on the level of individual functional groups. The authors viewed

that synthesizing DNAzymes in a straightforward design of modified nucleotides will

existing and achievable knowledge with the four basic nucleotides on the scale of

individual functional groups.

3. In vitro selection and study of active DNAzymes in the presence of spectroscopically

rich paramagnetic metal ions. The authors also recommended investigating

spectroscopically to obtain new DNAzymes that can specifically bind paramagnetic

metal ions such as iron, manganese, and nickel. It will expand the scope of DNAzyme

activity, allowing the use of a full range of spectroscopic techniques used with

metalloproteins and ribozymes like the electron magnetic resonance (EPR).

The journal is impressive because I got much knowledge that could apply to Biochemistry,

specifically on enzymes. This journal emphasizes the biochemical studies of DNAzymes, a

metalloenzymes class. When we say biochemical, it is more on the mechanism or the mode of

action of the DNAzymes, if it has interacted or bonded to different types of metal ions, which can

be monovalent, divalent, or trivalent, and even with modified nucleotides. On the other hand,

biophysical studies are much more focused on the structural characterization of the DNAzymes

using different analytical techniques such as NMR, x-ray crystallography, photocrosslinking, and

photocleavage. However, the structural determination of DNAzymes is comparatively

underexplored; hence, it has been a big recommendation to conduct more studies about this

because it can better understand the sequential assignment of DNAzymes. Biochemical and

biophysical coincides by focusing on the spectroscopic characterization of the metal-binding site

in DNAzymes by using different spectroscopic methods such as x-ray absorption spectroscopy

(XAS), fluorescent resonance energy transfer (FRET), circular dichroism (CD), and sensitized

luminescence.

This kind of study indeed provides a wealth of information on the metal-binding sites of

DNAzymes, and it has been observed that an increased application of this, especially in making

metal ion sensors and even for cellular detection. Future work about DNAzymes having an

established suite of techniques will enable widespread use in studying metalloproteins. I also

realized how innovative the methods and techniques had been used to gain much more profound

information about metal ions’ interactions with DNAzymes. As stated in the journal, the in vitro

selection methods using modified nucleotides and more innovative biochemical, structural, and

spectroscopic techniques will give us a much deeper understanding of the metal binding sites in
DNAzymes. Hence, it will provide broader and more improved applications of metalloenzymes

that will surely help both in the environmental metal ion sensors and cellular metal detection. But

educators like me will surely gain new knowledge that I can include in my lessons in Chemistry

and an innovative topic that I can impart to my students.