Determining rare state populations in nucleic acid ensembles

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Several features of nucleic acid behavior including RNA folding, protein-nucleic acid binding, and assembly of large nucleoprotein complexes (such as the ribosome or splicesosome) are dictated by the dynamic properties of nucleic acid molecules in solution. Understanding the properties of nucleic acids which influence their conformational landscape, and thereby their ability to engage in a multitude of functions, is an important biophysical challenge. While the methods of small angle X-ray scattering, Au-Au X-ray interferometry and nuclear magnetic resonance have enabled a characterization of the nucleic acid energy landscape, they do not allow for the characterization of low-populated states in the energy landscape. Low-populated states in nucleic acid energy landscapes play an important role because they represent intermediates in the molecules' folding pathway, and hence their identification and characterization could enable an understanding of higher order nucleic acid binding or assembly processes. In this work, a crosslinking approach has been employed to derive an understanding of low-populated states in the energy landscape of a nucleic acid. The crosslinking approach relies on using nucleic acid conjugated with a disulfide on one hand and a thiol modification on the other, which upon reaction result in the formation of nucleic acids crosslinked via a disulfide bridge. With this approach, the kinetics of disulfide bridge formation is measured and the populations in low-populated states, inferred. The crosslinking approach was applied to a number of nucleic acid motifs in order to provide a proof-of-principle that it can be employed to resolve differences in dynamics between them. The approach was extended to measure populations of molecules in low-populated states within nucleic acid ensembles, through estimations of an Effective Molarity (EM). The approach applied to a double-stranded DNA duplex showed that a large variation in EM for crosslink tether positions on the dsDNA duplex could be observed. When applied to double-stranded DNA duplexes linked by a polyethylene glycol (PEG) junction, relative populations within different subspaces of the molecule could be determined. The approach was also utilized to determine the dynamic circularization behavior of an RNA motif. The experiments described in this work indicate that the crosslink approach may indeed be useful to probe low-populated state populations in nucleic acid ensembles. It permits us to further our understanding of nucleic acid folding and higher order binding and assembly processes, since it opens us the possibility of isolating and characterizing low-populated states, which serve as intermediates in these processes.


Type of resource text
Form electronic; electronic resource; remote
Extent 1 online resource.
Publication date 2017
Issuance monographic
Language English


Associated with Gowrishankar, Seshadri
Associated with Stanford University, Department of Chemical Engineering.
Advisor Herschlag, Daniel
Advisor Khosla, Chaitan, 1964-
Advisor Spakowitz, Andrew James
Thesis advisor Herschlag, Daniel
Thesis advisor Khosla, Chaitan, 1964-
Thesis advisor Spakowitz, Andrew James


Genre Theses

Bibliographic information

Statement of responsibility Seshadri Gowrishankar.
Note Submitted to the Department of Chemical Engineering.
Thesis Thesis (Engineering)--Stanford University, 2017.
Location electronic resource

Access conditions

© 2017 by Seshadri Gowrishankar
This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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