# Directed Evolution of an Epitranscriptomic Toolkit

> **NIH NIH F32** · BROAD INSTITUTE, INC. · 2021 · $65,994

## Abstract

PROJECT SUMMARY/ABSTRACT
 The post-transcriptional modification of RNA is critical for RNA stability, trafficking, and translation into
proteins. In humans, disruption of the most prevalent RNA modification, N6-methyladenosine (m6A), has been
linked to diseases such as cancer. To better understand the role of m6A in disease pathogenesis, biologists
require robust methods for mapping m6A sites within RNA. Despite recent progress, existing methods require
numerous steps, lack selectivity and sensitivity, or demonstrate sequence bias. The long-term goal of this project
is to address these limitations by developing enzymes that can detect m6A and applying these tools for
epitranscriptomic analysis of cancer cells. Two strategies will be pursued in parallel: 1) Evolve reverse-
transcriptases, which polymerize DNA using RNA as a template, to incorporate a nucleotide “marker” into DNA
at m6A sites during reverse transcription of cellular RNA. This would facilitate direct readout of m6A without the
requirement for antibody-based pulldowns. 2) Engineer deaminases to mark m6A sites. Adenine deaminases
convert adenine (A) to inosine (I), which is read as guanine (G) by polymerases. Thus, engineering deaminases
to selectively deaminate m6A, rather than A, would result in a mark that can be located by RNA sequencing.
Finally, the evolved reverse transcriptase and deaminase platforms will be thoroughly biochemically
characterized and implemented for m6A sequencing of mammalian cells to establish the proposed platform. The
development and dissemination of these tools will have a broad impact by facilitating studies of m6A and its role
in human health.

## Key facts

- **NIH application ID:** 10312494
- **Project number:** 1F32GM143776-01
- **Recipient organization:** BROAD INSTITUTE, INC.
- **Principal Investigator:** Monica Neugebauer
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $65,994
- **Award type:** 1
- **Project period:** 2021-09-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10312494

## Citation

> US National Institutes of Health, RePORTER application 10312494, Directed Evolution of an Epitranscriptomic Toolkit (1F32GM143776-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10312494. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*