Studying RNA Using Single Molecule Fluorescence Resonance Energy Transfer

The intrinsic structure of RNA and in particular the dynamics of the same are of paramount importance for RNA function. Recent work, e.g., on riboswitches and ribosomes, shows the vast complexity of these dynamics. Consequently the interest in biochemical and biophysical methods to investigate these dynamics is rising. Here we describe the use of single molecule fluorescence resonance energy transfer (FRET) to investigate structural dynamics of individual immobilized RNA molecules. Such observations on the single molecule level allow the description of equilibria between structural populations, even down to transient subpopulations, which would otherwise been hidden in the ensemble average. This chapter starts with a brief introduction into FRET, followed by considerations on construct design, as well as dye choice and the layout of an instrumental setup used in our group. In an experimental part, RNA labeling methods are presented, as well as measurement chamber assembly, buffers that enhance dye performance, and actual sample preparation.