# Using Fitness Landscapes to assess RNA Structure-Function Relationships From Inside the Cell

> **NIH NIH R01** · BOSTON COLLEGE · 2021 · $391,250

## Abstract

Summary
RNA is an important therapeutic target, yet most RNA sequence-structure-function analysis is conducted in
vitro rather than in the context of the cell despite substantive differences between the two environments. The
objective of this proposal is to explore RNA sequence/function relationships within the cell using local fitness
landscapes. Our model systems for this work are bacterial RNA regulators, such as riboswitches, which are
antimicrobial targets and important biophysical models for studying RNA sequence-function relationships.
Based on previous studies in the literature and our own preliminary data, we hypothesize that in vitro
experiments imperfectly capture the sequence requirements required for biological function for many structured
RNAs. To address how in vitro measured parameters correspond with cellular expression and organismal
fitness, we plan to use three bacterial RNA regulators as model systems: the glycine riboswitch in Bacillus
subtilis, the pyrR regulator in Streptococcus pneumoniae, and an FMN riboswitch in S. pneumoniae. Our
specific aims are: 1) Determine which sequence changes to an RNA regulator impact organismal fitness and
how these may be altered under different conditions that include defined culture medium, the presence of
antibiotics, and a mouse infection model. 2) Establish the relationship between cellular gene expression profile
and organismal phenotype. 3) Assess how gene expression and organismal fitness correspond with in vitro
structure and function assays. We will achieve these aims by creating local fitness landscapes for each RNA
regulator wherein all single and most double mutants are created as a pool, and subsequently evaluated using
high-throughput sequencing as a final readout. The pools of variants will be appraised using a variety of
techniques including: organismal competitions in culture (B. subtilis and S. pneumoniae), in the presence of
targeting antibiotics (S. pneumoniae), and in a mouse infection model (S. pneumoniae); as well as microfluidic
encapsulation of microcolonies to enable FADS-based assessment of cellular gene expression and in vitro
transcription termination parameters. These high-throughput observations will be followed by assessment of a
subset of mutants to confirm the observed phenotypes and examine secondary structure both in vitro and
within the cell (SHAPE-MaP). Our proposal leverages organismal phenotypes we discovered in previous work
and is an innovative approach to assess RNA sequence-function relationships that cannot be assessed in any
other way. The expected outcome of the proposed work is the first mapping between RNA regulator sequence
and a range of variables including organismal phenotype, gene expression, and in vitro termination
characteristics. This contribution is significant because it will enable better interpretation of results from
traditional in vitro and gene expression assays to inform efforts to target RNA regulators with antibiotics and t...

## Key facts

- **NIH application ID:** 10141264
- **Project number:** 5R01GM134259-02
- **Recipient organization:** BOSTON COLLEGE
- **Principal Investigator:** Michelle Margaret Meyer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $391,250
- **Award type:** 5
- **Project period:** 2020-05-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141264

## Citation

> US National Institutes of Health, RePORTER application 10141264, Using Fitness Landscapes to assess RNA Structure-Function Relationships From Inside the Cell (5R01GM134259-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10141264. Licensed CC0.

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