Folding of RNA in the cell is not well understood nor has it been integrated into a cohesive mechanistic framework. The broad objectives of this proposal are to develop comprehensive molecular mechanisms for how functional RNAs fold in vivo and to relate these mechanisms to the evolutionary forces that help shape them. A comprehensive approach will be taken in which both the biophysical and evolutionary driving forces that give rise to RNA folding mechanism in vivo will be identified. The first specific aim will establish biophysical principles for in vivo RNA folding by examining the folding mechanisms of several naturally occurring riboswitches and ribozymes in both model cytoplasms and in cells. The second specific aim will elucidate evolutionary principles that guide RNA folding in vivo by testing the folding mechanisms of sequences that will emerge from several neutral drift selections. Thus, both naturally occurring and laboratory-evolved functional RNAs will be examined, with an overall goal of elucidating general principles for RNA folding in the cell. The research involves developing a series of model cytoplasms and testing the folding cooperativity and kinetics of RNAs in these. In addition, studies of folding mechanism will be conducted directly in eukaryotic and prokaryotic cells using several novel approaches. The role of evolutionary forces in shaping RNA folding landscapes in vivo will be studied by conducting several neutral drift selections. Members of these libraries will be assessed for folding thermodynamics and kinetics. The ability of cooperatively folding RNAs to adapt to selective pressures will also be assessed. Methods to be applied throughout this research include high-throughput calorimetry; CD and UV-detected thermal denaturation; rapid kinetics; SAXS; and the expression, structure mapping, and live cell imaging of RNAs in various cells. Data throughout the research will be modeled by several theoretical and computational approaches, which will be used both to help understand folding behavior and to refine experiments. Because new insights into RNA folding dynamics and adaptation should be revealed, the results should broadly influence many different health- related projects. The findings may make it possible to rationally engineer RNA therapeutics with different in vivo stabilities, and they may lead to new insights into the relationship between genotype and phenotype in viruses.

Public Health Relevance

Understanding the interplay between secondary and tertiary structure in in vivo RNA folding cooperativity may make it possible to rationally engineer RNAs with different in vivo stabilities, which could have important applications in the development of therapeutics with different half-lives. Moreover, the findings may lead to new relationships between genotype and phenotype in viruses. Finally, the findings herein may unearth folding principles that are general and apply to all functional polymers (e.g. RNA and DNA aptamers and enzymes, and proteins), potentially attesting to the universality of evolutionary forces in shaping RNA folding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM110237-03
Application #
9060969
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter
Project Start
2014-08-01
Project End
2018-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
Leamy, Kathleen A; Yennawar, Neela H; Bevilacqua, Philip C (2018) Molecular Mechanism for Folding Cooperativity of Functional RNAs in Living Organisms. Biochemistry 57:2994-3002
Yamagami, Ryota; Bingaman, Jamie L; Frankel, Erica A et al. (2018) Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis. Nat Commun 9:2149
Leamy, Kathleen A; Yennawar, Neela H; Bevilacqua, Philip C (2017) Cooperative RNA Folding under Cellular Conditions Arises From Both Tertiary Structure Stabilization and Secondary Structure Destabilization. Biochemistry 56:3422-3433
Bingaman, Jamie L; Messina, Kyle J; Bevilacqua, Philip C (2017) Probing fast ribozyme reactions under biological conditions with rapid quench-flow kinetics. Methods 120:125-134
Leamy, Kathleen A; Assmann, Sarah M; Mathews, David H et al. (2016) Bridging the gap between in vitro and in vivo RNA folding. Q Rev Biophys 49:e10
Bingaman, Jamie L; Frankel, Erica A; Hull, Chelsea M et al. (2016) Eliminating blurry bands in gels with a simple cost-effective repair to the gel cassette. RNA 22:1929-1930
Hull, Chelsea M; Anmangandla, Ananya; Bevilacqua, Philip C (2016) Bacterial Riboswitches and Ribozymes Potently Activate the Human Innate Immune Sensor PKR. ACS Chem Biol 11:1118-27
Hull, Chelsea M; Bevilacqua, Philip C (2016) Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR. Acc Chem Res 49:1242-9
Hull, Chelsea M; Bevilacqua, Philip C (2015) Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA. J Mol Biol 427:3501-3515