Probing RNA conformational dynamics and heterogeneity using femtosecond time-resolved fluorescence spectroscopy

RNA structures are very dynamic and the dynamic motions result in a heterogeneous conformational ensemble. It is crucial to illustrate the role of conformational dynamics in RNA function. A variety of spectroscopic methods have been used to investigate the dynamic aspects of RNA structures. Recently, ultrafast time-resolved spectroscopy, a well-established technique, has been introduced as a new tool in this field. With femtosecond time-resolution, one can resolve the heterogeneous nature of RNA conformational ensemble quantitatively, detect and characterize minor unusual conformations, and capture folding events that may occur on a fast timescale. Here, we review the technical aspects of using an ultrafast fluorescence up-conversion technique to probe the heterogeneous base stacking patterns in RNA motifs and dynamic base motions that allow sampling of multiple states.