Using analytical ultracentrifugation (AUC) to measure global conformational changes accompanying equilibrium tertiary folding of RNA molecules

Abstract

Analytical ultracentrifugation (AUC) is a powerful technique to determine the global conformational changes in RNA molecules mediated by cations or small molecule ligands. Although most of the developments in the field of AUC have been centered on studies involving protein molecules, the experimental methods as well as the analytical approaches have been successfully adapted and applied to the study of a variety of RNA molecules ranging from small riboswitches to large ribozymes. Most often AUC studies are performed in conjunction with other structural probing techniques that provide complementary information on local changes in the solvent accessibilities at specific regions within RNA molecules. This chapter provides a brief theoretical background, working knowledge of instrumentation, practical considerations for experimental setup, and guidelines for data analysis procedures to enable the design, execution, and interpretation of sedimentation velocity experiments that detect changes in the global dimensions of an RNA molecule during its equilibrium folding.