# High-throughput screening and structure-guided optimization of oligonucleotides for site-directed RNA editing by ADARs.

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2023 · $332,039

## Abstract

Project Summary
The largest class of genetic alterations that cause disease are single point mutations. Most of
these disease-causing errors can be remedied if a specific adenosine is changed to guanosine
on the RNA transcript. This proposal aims to repurpose an RNA editing enzyme and direct it to
selectively edit targeted adenosines in mRNA to treat genetic disorders. The RNA editing enzyme
Adenosine Deaminase acting on RNA (ADAR) can convert adenosine to inosine (A-to-I) by
catalyzing a deamination reaction on the nucleobase. Inosine is read as guanosine by the cellular
translation machinery providing the ability to alter codons in mRNA. This proposal will focus on
selectively editing disease-causing nonsense mutations, by directing ADAR to edit the adenosine
in the stop codon thus allowing the transcript to continue translation, producing functional full-
length protein. Because ADARs selectively edit adenosines in regions of dsRNA, disease-causing
nonsense mutations can be selectively targeted by furnishing an appropriate guide
oligonucleotide to create a dsRNA substrate. Canonical Watson-Crick complementarity of duplex
RNA does not produce efficient substrates for ADARs, making it challenging to design effective
guide oligonucleotides to target specific nonsense mutations. A high-throughput assay is
proposed to search all sequence space of guide RNA oligonucleotides to identify lead sequences
displaying high editing efficiency of the targeted nonsense transcript using endogenous ADARs.
These lead sequences can be further optimized by structure-guided rational design methods. The
lab has significant experience in determining ADAR-RNA structures to atomic resolution and
leveraging this knowledge to improve editing efficiency. Both X-ray crystallography and Cryo-EM
techniques are proposed for ADAR1 or ADAR2 complexed with dsRNA of the lead sequence
bound to its targeted mRNA segment. These structures will provide the basis for rational design
adjustments to develop nucleotide analogs to incorporate into guide oligonucleotides that can
fashion structural features for improved editing and increased metabolic stability. This method of
site-directed RNA editing (SDRE) to treat genetic disorders offers many advantages over current
editing tools, which often require addition of sizable proteins (e.g., CRISPR/Cas). When fully
developed, this method would permit the simple administration shorter oligonucleotides allowing
the cell’s endogenous ADARs to recode the nonsense mutation to treat many genetic disorders.

## Key facts

- **NIH application ID:** 10636547
- **Project number:** 1R01GM149799-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** ANDREW J FISHER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $332,039
- **Award type:** 1
- **Project period:** 2023-04-01 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10636547

## Citation

> US National Institutes of Health, RePORTER application 10636547, High-throughput screening and structure-guided optimization of oligonucleotides for site-directed RNA editing by ADARs. (1R01GM149799-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10636547. Licensed CC0.

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