Spectroscopic Probes of RNA Structure and Dynamics

A single technique is insufficient to characterize the properties of an RNA molecule so this Guide provides advice and suggestions for use of several spectroscopic methods applied to RNA. It begins with a discussion of design features to synthesize a suitable molecule for study, assuming that the reader is familiar with in vitro enzymatic and chemical synthesis of RNA. With the RNA in hand, the application of UV melting studies to characterize its folding/unfolding transitions is presented, followed by instructions for fluorescence experiments to augment the UV data. Since RNAs are flexible molecules, it is important to characterize their mobility, and so time-resolved fluorescence data are analyzed. A brief exposition of the power of NMR spectroscopy to identify ion-binding sites is provided. RNA examples are described for each method to give a perspective on what can and cannot be learned.