Date of Award
1-1-2019
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
College/School/Department
Department of Biological Sciences
Content Description
1 online resource (xiv, 202 pages) : illustrations (some color)
Dissertation/Thesis Chair
Pan T.X. Li
Committee Members
Dan Fabris, Ben Szaro, Alan Chen
Keywords
RNA, Electrospray ionization mass spectrometry, Protein folding, HIV (Viruses), RNA viruses
Subject Categories
Biology
Abstract
RNA’s diverse gene regulatory functions are tied to its ability to adopt and rearrange between an ensemble of three-dimensional structures. This concept is illustrated by the process of genome dimerization in HIV-1 and other retroviruses, which is mediated by the dimerization initiation site (DIS) of viral RNA. This essential stem-loop domain establishes a metastable kissing complex (KC) intermediate that seeds the structural rearrangements necessary to stabilize genome dimerization. Most approaches applied to study RNA structure provide us with a snapshot of RNA at equilibrium, leaving key details on dynamics concealed. This thesis explored the merits of nanospray ionization mass spectrometry (nanospray MS) as an analytical platform for the elucidation of RNA structural rearrangements. We combined solution thermal melting and nanospray MS to extend analysis of samples beyond room temperature. We employed selected oligo-deoxynucleotide models to establish experimental conditions for MS analysis, and delineated analysis approaches to study dimerization equilibria. Furthermore, we expanded measurements towards non-equilibrium, by developing an RNA exchange approach. We were able to capture previously inaccessible information about RNA KC association and dissociation kinetics. Finally, we combined all our tools to track the rearrangement of the HIV-1 DIS kissing complex into an extended duplex (ED), demonstrating that nanospray MS can be effectively employed to study structural rearrangements of nucleic acids. Our approach enabled a very detailed, ensemble-level characterization of the rearrangement process. This dissertation highlighted the flexibility and robustness of this platform for the investigation of RNA structural rearrangements.
Recommended Citation
Toro, Botros, "Examining the mechanisms of nucleic acid structural rearrangements using nanospray ionization mass spectrometry" (2019). Legacy Theses & Dissertations (2009 - 2024). 2398.
https://scholarsarchive.library.albany.edu/legacy-etd/2398