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

RNA molecules can form secondary and tertiary structures that determine their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. A limiting factor, however, is that only population averages can be obtained, since each read is an independent measurement. In addition, no information about structural heterogeneity across molecules or dependencies within each molecule is accessible.

Here, we present Single Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules.

Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. In addition to revealing known structural features of mRNAs, SMS-seq shows compartmentalization of structural dependencies across CDSs and 3′UTRs. Finally, we show that SMS-seq can capture structural breathing, tertiary interactions and dynamics of riboswitch ligand binding.