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  4. STRUCTURAL STUDIES OF VIRAL RNA THROUGH SMALL ANGLE X-RAY SCATTERING AND CONTRAST VARIATION

STRUCTURAL STUDIES OF VIRAL RNA THROUGH SMALL ANGLE X-RAY SCATTERING AND CONTRAST VARIATION

File(s)
SanEmeterio_cornellgrad_0058F_13029.pdf (13.53 MB)
Permanent Link(s)
https://doi.org/10.7298/65r9-q584
https://hdl.handle.net/1813/111782
Collections
Cornell Theses and Dissertations
Author
San Emeterio, Josue
Abstract

RNA viruses are pathogens that affect human life. Their RNA genome does not only hold genetic information; it is a functional element capable of regulating transcription and assembly. Understanding the structure of viral RNA can accelerate drug discovery and help develop antiviral therapeutics. We are interested in two mechanisms of viral RNA: The -1 programmed ribosomal frameshifting pseudoknot of SARS-CoV-2 and the RNA-mediated viral assembly of small +ssRNA viruses. The RNA structures in these two processes are not yet fully understood. We study these structures using small angle x-ray scattering (SAXS) and contrast variation SAXS (CV-SAXS). SAXS can elucidate the structure of viral RNA in solution and resolve dynamics. Contrast variation SAXS (CV-SAXS) preferentially monitors the structure of RNA as viruses assemble. First, we use size exclusion coupled SAXS (SEC-SAXS) to obtain the solution scattering of the pseudoknot, and we compare it to the computed scattering from cryo-EM and crystallography structures. We then use SAXS-driven MD (Molecular Dynamics) to obtain models that better fit the SAXS data. We apply this same approach to a pseudoknot mutant that does not frameshift. We comment on the structural differences between the wild type and mutant pseudoknot models and how these differences affect frameshifting. We then provide protocols for performing a CV-SAXS experiment. Later, we examine some of the non-idealities of the CV-SAXS method that emerge from the hydration shell of biomolecules. Finally, we apply CV-SAXS to study bacteriophage MS2 and demonstrate that the CV-SAXS method can be used on an entire virus. On this basis, we propose that CV-SAXS can be used to monitor the assembly of viruses from their early stages to completion.

Description
198 pages
Date Issued
2022-05
Keywords
Contrast Variation
•
CV-SAXS
•
Programmed Ribosomal Frameshifting
•
RNA Virus
•
SARS-CoV-2
•
SAXS
Committee Chair
Pollack, Lois
Committee Member
Gruner, Sol Michael
Stroock, Abraham Duncan
Degree Discipline
Applied Physics
Degree Name
Ph. D., Applied Physics
Degree Level
Doctor of Philosophy
Type
dissertation or thesis
Link(s) to Catalog Record
https://newcatalog.library.cornell.edu/catalog/15529871

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