Sequence Dependent RNA UNCG and DNA TNCG Tetraloop Hairpin Melting and Refolding Dynamics Using Temperature-Jump Infrared Spectroscopy

Temperature-jump Infrared spectroscopy (T-jump IR) is a label-free method that provides access to non-equilibrium dynamic processes on timescales of nanoseconds to milliseconds, capturing changes in vibrational modes that indicate changes in molecular structure. This thesis outlines the development of T-jump IR methodologies to study RNA UNCG tetraloop hairpins, an RNA motif important for proper folding of its tertiary structure and stability. To gain insight into the structural basis for their relative behaviour, analogous DNA hairpins were also studied. Chapter 3 presents a T-jump methodology to measure RNA and DNA melting and refolding dynamics in a single measurement for the first time. This established that RNA and DNA hairpins melt on different timescales. Their refolding times however were similar, with the formation of base-stacks the rate limiting step for both. In Chapter 4, the influences of the stem sequence on the RNA and DNA hairpin dynamics are investigated by introducing an AT pair into the stem sequence to provide site-specific information. By tracking the dynamic changes at particular positions in the stem, the pathways of melting and refolding can be established by comparison. These experiments determined the RNA and DNA tetraloops melt and refold via different intermediate conformations. Chapter 5 extends the study of the UNCG tetraloop dynamics using Two-dimensional infrared spectroscopy (2D-IR) and T-jump 2D-IR in order to observe the changes in the relationships of the vibrational modes during the melting dynamics. By doing this a greater level of structural detail can be obtained, allowing more precise determination of the conformational dynamics over the melting process. This work suggested DNA melting follows a more multistate transition than RNA, due to greater flexibility in the stem.