# DNAzymes for Site-Specific DNA and RNA Nucleobase Modification

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2024 · $290,976

## Abstract

Project Summary
 This Focused Technology Research and Development proposal addresses the urgent need for new tools to
perform in vitro site-specific modification of DNA and RNA nucleobases of long nucleic acid substrates. The
biochemistry, biophysics, and biology of DNA and RNA are heavily influenced by particular nucleobase
modifications. However, studies of these modifications are often limited by the ability to synthesize the modified
nucleic acids. Therefore, innovative new technologies are required to circumvent the limitations of the existing
chemical and enzymatic modification approaches.
 DNAzymes (deoxyribozymes) are artificial DNA sequences with enzymatic function, analogous to protein
enzymes as catalytic sequences of amino acids. Although DNAzymes are unknown in nature, they can be
identified de novo in the laboratory by starting with random DNA sequence pools and performing in vitro selection
for the desired enzymatic activity. DNAzymes are operationally simple to obtain and use: they are readily
prepared commercially by solid-phase synthesis, and they easily modify their substrates in simple aqueous
incubation conditions with standard buffers and salts. DNAzymes have been identified for a growing range of
catalytic activities, but most applications of DNAzymes are limited to the two long-known reactions of RNA
cleavage and hemin-dependent peroxidation, which are unrelated to DNA and RNA nucleobase modification.
 In the proposed studies, DNAzymes will be identified for a qualitatitvely new approach to site-specific in vitro
modification of DNA and RNA nucleobases, by either N-acylation or (via reductive amination) N-alkylation at N4
of cytosine, N2 of guanine, and N6 of adenine. Rather than relying on natural protein enzymes, either as found
in nature or evolved by directed evolution, entirely new DNAzymes whose sequences are unconstrained by
natural evolutionary history will be identified by in vitro selection.
 Three specific aims are proposed. In Aim 1, in vitro selection will be used to identify DNAzymes for N-
acylation of C, G, and A nucleobase amines in DNA and RNA substrates. In parallel Aim 2, in vitro selection will
be used to identify DNAzymes for N-alkylation by reductive amination of C, G, and A nucleobase amines in DNA
and RNA substrates. In both of these aims, the electrophilic reaction partner will be either an oligonucleotide or
a small molecule, as a tunably fluorinated aryl ester for the acyl donor in Aim 1, and as an aromatic or aliphatic
aldehyde, or activated imine variant thereof, for the reductive amination partner in Aim 2. In Aim 3, the DNAzymes
from Aims 1 and 2 will be characterized with regard to their substrate sequence scope and refined by reselection
for improved function, to facilitate practical use as an innovative new technology for site-specific DNA and RNA
nucleobase modification.
 By the conclusion of the proposed efforts, we will have established DNAzymes as an important new
technology for in vi...

## Key facts

- **NIH application ID:** 10901830
- **Project number:** 5R01GM149566-02
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Scott K Silverman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $290,976
- **Award type:** 5
- **Project period:** 2023-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10901830

## Citation

> US National Institutes of Health, RePORTER application 10901830, DNAzymes for Site-Specific DNA and RNA Nucleobase Modification (5R01GM149566-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10901830. Licensed CC0.

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