# Mapping the sequence landscape of RNA structure, dynamics and protein interactions using high-throughput single-molecule FRET

> **NIH NIH R35** · UNIVERSITY OF OREGON · 2022 · $353,023

## Abstract

Project Summary
 RNA plays a central role in nearly every biological process, acting in turn as a messenger,
catalyst, signaling molecule and more. Many of these roles require RNA to fold into specific
structures, and a given RNA species can often adopt multiple structures that modulate properties
such as protein interactions, ligand binding and catalytic activity. Research in the Widom Lab
focuses on developing and applying new spectroscopic methods to study RNA structure and
dynamics. We combine bulk, single-molecule and ultrafast spectroscopy in order to obtain a
comprehensive picture of RNA folding and interactions over length-scales from Angstroms to
microns and time-scales as short as picoseconds. We are currently using these methods to study
RNA-protein interactions during pre-messenger RNA (pre-mRNA) splicing, the process in which
segments of RNA that do not code for protein are excised, and ligand binding by riboswitches,
which regulate gene expression in bacteria.
 Single-molecule measurements offer a unique window into the heterogeneous and
dynamic nature of RNA folding, and ultrafast spectroscopy probes very rapid processes that are
inaccessible by other means. However, these are notably low-throughput techniques, with
measurements typically being performed on only a single RNA sequence at a time. An entirely
new class of scientific questions could be addressed if these techniques could be applied in a
high-throughput manner. We will bring this goal to realization by developing a process for
performing single-molecule fluorescence measurements on hundreds of different sequences
simultaneously. A library of RNA sequences will be prepared containing random bases at sites of
interest and the mixture will be subjected to single-molecule fluorescence measurements to
monitor structural rearrangements and response to stimuli in real time. Each molecule will then
be sequenced in situ in order to determine what sequence gave rise to its single-molecule signal.
Our goals for the next 5 years are to optimize this method and to use it to answer questions that
can only be addressed via high-throughput approaches. We will investigate the mechanistic
impacts of key sequences on pre-mRNA splicing, including variable sequences that fine-tune
splicing and conserved sequences that lead to disease when disrupted. We will also measure the
folding thermodynamics and kinetics of hundreds of RNA sequences and use the results to
benchmark and improve RNA structure prediction algorithms. This research program will greatly
increase the throughput of single-molecule fluorescence measurements, enabling detailed
biophysical insights to be achieved rapidly across a vast sequence space.

## Key facts

- **NIH application ID:** 10500676
- **Project number:** 1R35GM147229-01
- **Recipient organization:** UNIVERSITY OF OREGON
- **Principal Investigator:** Julia Reed Widom
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $353,023
- **Award type:** 1
- **Project period:** 2022-09-21 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10500676

## Citation

> US National Institutes of Health, RePORTER application 10500676, Mapping the sequence landscape of RNA structure, dynamics and protein interactions using high-throughput single-molecule FRET (1R35GM147229-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10500676. Licensed CC0.

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