Single molecule structure sequencing reveals RNA structural dependencies, breathing and ensembles

We develop a method, Single Molecule Structure sequencing (SMS-seq), that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual RNA molecules. RNA is probed using diethyl pyrocarbonate, which modifies single-stranded adenosine residues. The RNA was sequenced using Oxford nanopore sequencing technology using MinION Flow Cell (R9.4). Modified residues were used as proxy to determine the accessibility of a region. SMS-seq reveals known structural features of yeast in vitro folded mRNAs and non-coding RNAs. It shows compartmentalization of structural dependencies in CDSs and 3'UTRs. SMS-seq captures RNA structural breathing, tertiary interactions and dynamics of riboswitch upon ligand binding.