# Probing the Transcriptome with Multifunctional Acylation Chemistry

> **NIH NIH R01** · STANFORD UNIVERSITY · 2021 · $319,854

## Abstract

Recent studies from many labs have uncovered great complexity in cellular RNA biology, and critically
important connections of RNA to human health. It is becoming increasingly apparent that RNA biology, like
protein biology, is not merely peripheral, but rather central to cellular phenotypes and pathologies.
Unfortunately, methods for study of RNA, such as tools for functionalization, labeling and control, lag well
behind those used widely for proteins.
 Preliminary experiments have established the promise of a suite of novel molecular strategies for study of
RNAs, based on multifunctional acylating agents that react at the 2'-OH group. This started with the
development of the first cell-permeable acylating agents, based on a nicotinyl scaffold, that react with
accessible 2'-OH groups in RNAs. These reagents allow unprecedented measurement of RNA structure and
protein-RNA interactions in vivo at nucleotide resolution. In unpublished work, studies have shown that an
azide functional handle can be employed on these acylating scaffolds to enable mild, bioorthogonal reversal
of the acylation by Staudinger reduction. Excitingly, experiments show that this acylation/deacylation strategy
can be used to block and initiate hybridization of RNA. Moreover, the data establishes that a label can be
incorporated into such an acylating agent, enabling one-step, reversible fluorescent labeling of native RNA.
 These preliminary experiments suggest a suite of new acylating reagents as tools to isolate, immobilize,
label, and analyze RNAs, and a range of molecular strategies to control their biological activities with
chemical or optical signals. During the term of this project, the development of reversible protecting reagents
for stabilizing and capturing RNAs from biological samples are proposed. Reagents for covalent delivery and
release of RNAs into cells are also described. Further, new fluorescent acylating agents and methods will be
developed and employed to measure protein-RNA interactions. Finally, a novel range of unprecedented
chemical caging and release strategies will be developed for controlling biological function of RNAs in living
systems, enabling initiation of mRNA expression, RNA folding, and gene editing in time and space.
 This work is significant because it will develop enabling molecular technologies that will greatly enhance
the study of RNA biology and biomedicine. This new premise of multifunctional acylation will lead to universal
and easy-to-use reagents that will markedly improve the isolation, analysis, delivery, and control of RNAs for
researchers worldwide. Unlike previous methods, these reagents will function with large and native RNAs,
and are simple enough that non-chemists can apply them. The research program is innovative because it
develops a suite of new molecular probes and novel molecular strategies, making use of the concept of
reversible labeling and functionalization of RNA via new selective bond-forming and –breaking st...

## Key facts

- **NIH application ID:** 10147102
- **Project number:** 5R01GM127295-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** ERIC T. KOOL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $319,854
- **Award type:** 5
- **Project period:** 2018-07-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10147102

## Citation

> US National Institutes of Health, RePORTER application 10147102, Probing the Transcriptome with Multifunctional Acylation Chemistry (5R01GM127295-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10147102. Licensed CC0.

---

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