Sensitive detection of structural features and rearrangements in long, structured RNA molecules

Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suited for a given application. Here, using a long, highly structured viral RNA as an example (the positive strand genome of Hepatitis C Virus), we present a semi-comprehensive analysis and describe the major approaches for analyzing the architecture of RNA that is modified with structure-sensitive probes. Additionally, we present an updated method for generating in vitro transcribed and folded RNA that maintains native secondary structures in long RNA molecules. We anticipate that the methods described here will streamline the use of current approaches and help investigators who are unfamiliar with structure probing, obviating the need for time-consuming and expensive optimization.