Use of ¹⁹F NMR methods to probe conformational heterogeneity and dynamics of exchange in functional RNA molecules.

Abstract

Functional RNA molecules are often very plastic and often undergo changes in base-pairing patterns to achieve alternative secondary and tertiary conformations associated with their roles in multiple events in gene expression. Solution NMR techniques are an excellent tool for the analysis of conformational heterogeneity and dynamic exchange. In this work, we measure the rates associated with spontaneous interconversion between major conformers in folded RNA sequences by use of a (19)F-(19)F EXSY NMR experiment, taking advantage of RNA samples carrying a single 5-(19)F-pyrimidine label. We first utilize this approach to determine kinetic exchange rates between conformers in a model RNA stem loop capable of adopting two conformations. We then probe the dynamics of conformational rearrangements in a larger RNA construct, the U2-U6 snRNA complex of the human spliceosome. In the case of the U2-U6 snRNA complex, such a rearrangement in the context of the intact spliceosome may have critical implications in splicing activity.