# RNA folding and catalysis at the interface of biophysics and genomics

> **NIH NIH R35** · PENNSYLVANIA STATE UNIVERSITY, THE · 2021 · $381,333

## Abstract

Abstract
The Bevilacqua lab has contributed to the field of RNA biology through two disparate approaches. One is
rigorous ribozyme mechanism and the other is discovery-based RNA structural genomics. A major goal of this
proposal is to bridge these two areas to discover new RNA biology and to characterize it at the molecular level.
This proposal advances a set of testable hypotheses on RNA folding and catalysis, as well as proposes new
technologies to enable discovery of novel RNA biology. Conservation of mechanistic strategies suggests ways
in which small ribozyme self-cleavage is activated by two unique catalytic strategies. These strategies lead to
specific changes in the hydrogen bonding status of the 2′OH that could lower its pKa to facilitate deprotonation.
Kinetics experiments, pKa measurements, and calculations will be implemented and conducted on a diverse
set of small ribozymes, as well as mechanistically related protein enzymes. In addition, cryoEM approaches to
solve structures of small, unmodified ribozymes involving preparation of nano-objects structures and rapid
vitrification will be pioneered. Techniques for cryoEM developed on small ribozymes will be applied to small
RNA and RNP complexes to expand the impact of cryoEM on the RNA field. Evidence is provided for a third
catalytic strategy, which is buffer catalysis in ribozymes, and a suite of experiments is proposed to test this.
Buffer catalysis could help explain how diverse RNA enzymes work including large ribozymes. We also seek
to understand RNA folding in vivo. Methods will be developed to measure RNA folding prediction rules under
in vivo-like and in vivo conditions. Complex artificial cytoplasms will be developed and used to measure binding
between a fluorescently labeled RNA and its unlabeled complement. Titrations in such `messy systems' will be
accomplished on a qPCR plate reader as a function of temperature to provide van't Hoff parameters.
Prediction rules for RNA folding will also be measured in vivo and genome-wide by applying Structure-seq to
Bacillus subtilis growing at different temperatures. The prediction rules should improve RNA folding prediction
under in vivo conditions. An inverse relationship between RNA and protein structure will be pursued by
measuring ribosome profiling on select proteins. Overall, synergy between mechanistic and genomic
approaches will be developed on multiple levels and includes pioneering techniques for detecting charged
bases for mechanistic studies and applying them genome wide. In addition, weakly chelated in vivo-like Mg2+
conditions provide an ideal system for investigating stimulation of RNA catalysis and folding. Finally, RNA
prediction rules developed in vivo will help describe the folding and function of catalytic RNAs. Computational
approaches play a key role. They aid prediction of RNA structure from sequence with new prediction rules,
help design cooperatively folding RNAs from structural descriptors, and allow testing o...

## Key facts

- **NIH application ID:** 10142493
- **Project number:** 5R35GM127064-04
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** PHILIP C BEVILACQUA
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $381,333
- **Award type:** 5
- **Project period:** 2018-05-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10142493

## Citation

> US National Institutes of Health, RePORTER application 10142493, RNA folding and catalysis at the interface of biophysics and genomics (5R35GM127064-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10142493. Licensed CC0.

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