Structural Studies Of Model Rna Systems In Solution Using F{\"O}Rster Resonance Energy Transfer

Abstract

New biological roles for RNA are continually being discovered, reinforcing the importance of understanding the fundamental forces governing RNA structure. Open questions remain about the structure of RNA because of the complex interplay between base pairing, base stacking, and counterion-mediated interac¨ tions. Forster resonance energy transfer (FRET) provides a powerful tool for measuring RNA conformational changes in solution. In this work we present three structural studies of model RNA systems using FRET. In the first two studies, we use single-molecule FRET to measure the subtle conformational changes on model single- and double-stranded RNA junctions induced by salt concentration. For single-stranded junctions at physiological salt concentrations both helix-helix repulsion and junction sequence are important factors determining RNA conformation. Furthermore, the sequence dependence of double stranded junctions is particularly important in solutions containing Mg2+ ions. In the third study, we measure sub-millisecond conformational changes of a small independently folding RNA induced by a jump in Mg2+ concentration in a microfluidic mixer. We show that mutations within a specific ion binding site can alter the folding rates of a small RNA sub-domain. We propose that understanding how subtle changes in ion concentration and RNA sequence can bias overall structure is necessary for a full picture of RNA folding.