Solvent Kinetic Isotope Effects on 2'-Hydroxyl Acylation of RNA

Abstract

RNA can fold into complex tertiary structures, much like proteins, and exhibits significant conformational dynamics that are a central facet of its functions. Two limiting types of RNA motions occur: equilibrium fluctuations and induced conformational changes. Equilibrium fluctuations are spontaneous transitions between conformers along the RNA free energy landscape and conformational transitions occur when the free energy landscape is perturbed to generate new minima and change the energy barriers between minima. Surprisingly, little experimental data are available for equilibrium fluctuations for even simple RNA, and even less is known about the driving forces for conformational transitions in large RNAs. In part this is due to limiting technologies. One technology, SHAPE, has overcome many of the limitations for studying large RNAs. SHAPE, for selective 2′-hydroxyl acylation analyzed by primer extension, has revolutionized RNA secondary structure prediction and provides a transformative experimental approach to investigate nucleotide specific equilibrium fluctuations and conformations. SHAPE reactivity is governed primarily by nucleotide flexibility, which in turn is governed by secondary and tertiary structure constraints. In practice, SHAPE provides nucleotide level resolution of RNA structure and dynamics in RNA of any size. We hypothesize that the solvent isotope effect on SHAPE chemistry will allow insights into the roles of hydrogen bonding and solvation on RNA structure and dynamics. Here, we describe the initial experiments and theoretic basis for interpreting this solvent isotope effect.