Probing RNA structure and dynamics using nanopore and next generation sequencing

It has become increasingly evident that the structures RNAs adopt are conformationally dynamic; the various structured states that RNAs sample govern their interactions with other nucleic acids, proteins, and ligands to regulate a myriad of biological processes. Although several biophysical approaches have been developed and used to study the dynamic landscape of structured RNAs, technical limitations have limited their application to all classes of RNA due to variable size and flexibility. Recent advances combining chemical probing experiments with next-generation- and direct sequencing have emerged as an alternative approach to exploring the conformational dynamics of RNA. In this review, we provide a methodological overview of the sequencing-based techniques used to study RNA conformational dynamics. We discuss how different techniques have enabled us to better understand the propensity of RNAs from a variety of different classes to sample multiple conformational states. Finally, we present examples of the ways these techniques have reshaped how we think about RNA structure.