Date of Award

1-1-2014

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Nanoscale Science and Engineering

Program

Nanoscale Engineering

Content Description

1 online resource (vi, 177 pages) : color illustrations.

Dissertation/Thesis Chair

Pan TX Li

Committee Members

Daniele Fabris, Nathaniel Cady, Thomas Begley

Keywords

Kissing complex, Optical tweezers, RNA folding, Single molecule, Thermodynamics, Trinucleotide repeats, RNA, Protein folding

Subject Categories

Biochemistry | Biophysics | Physics

Abstract

RNA folding is the process whereby a single stranded RNA molecule assumes its three-dimensional functional conformation. Along with the protein folding problem, the RNA folding problem remains as one of the great unsolved problems in biophysics. Generally RNA folding occurs in a hierarchical manner whereby the sequence of an RNA (primary structure) determines which regions will form helical segments (secondary structure) before further rearrangement and base pairing of secondary structure motifs (tertiary structure). Due to the intimate connection between structure and function within molecular biology, increased familiarity with the thermodynamic and kinetic factors that govern RNA folding will permit the decryption of complex regulatory pathways in the cell. RNA folding however, occurs within a convoluted environment and is influenced by a variety of factors, some of which include salt, temperature, pH, and excluded volume effects. Dissecting the contributions of these factors to RNA folding will help answer key questions about the nature of RNA folding such as: How does temperature affect RNA structure? What dictates RNA's ability to alternatively fold? What are the folding energetics for RNA tertiary interactions? In this work we attempt to address these questions from a single molecule perspective, comparing results to ensemble measurements where appropriate. Using optical tweezers we can apply force to unfold a single RNA molecule from its 5'- and 3'-ends while simultaneously measuring the extension of the RNA molecule. This permits the observation and nanomanipulation of folding pathways in real-time. Furthermore, we employ thermodynamic and kinetic approaches to obtain free energies and rate constants of RNA folding. Single molecule methods such as mechanical unfolding represent a valuable technique which can reinforce the results of bulk biochemical assays while simultaneously elucidating the information-rich dynamics of individual molecular folding trajectories.

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