Temperature-Controlled, Single-Molecule Kinetic Investigations of Nucleic Acid Folding Motifs

Nucleic acids serve many biological functions beyond information storage. These functions depend on the ability of a nucleic acid to adopt a specific spatial configuration in a process called folding. The folding of each nucleic acid sequence is unique, but there exist common folding patterns, motifs, that are shared by many nucleic acids. Folding typically occurs through a stepwise process in which motifs in a nucleic acid fold independently of each other before interacting to form higher order structures. The purpose of this dissertation is to provide a detailed account of the kinetic and thermodynamic properties of select motifs to give greater insight into how nucleic acids fold into functional forms.

The primary tool used in this work is single-molecule fluorescence resonance energy transfer (smFRET), which permits the observation of nucleic acid conformational dynamics in real-time and the concomitant determination of the first-order rate constants for folding and unfolding. Furthermore, performing kinetic measurements under controlled temperature enables the evaluation of the thermodynamic properties (e.g., enthalpy and entropy) of the folded and unfolded states, as well as of the transition state along the folding pathway. The nucleic acid motifs surveyed in this work are a DNA hairpin, an RNA tetraloop-tetraloop receptor, and an intramolecular DNA G-quadruplex. Temperature-controlled smFRET experiments on these motifs are used to probe the mechanism of motif folding, with a focus on the nature of charged ligand uptake during folding. Furthermore, the smFRET approach for studying conformational dynamics has been developed herein to expand the range of measurable rate constants and to obtain higher order thermodynamic information on nucleic acids.