Transcriptome-wide RNA structure probing with temporal resolution

Reactive small-molecule probes are widely used for RNA structure probing, however current approaches largely measure average RNA transcript dynamics and do not resolve structural differences that occur during folding or transcript maturation. Here, we present SNIPER-seq, an RNA structure probing method relying upon metabolic labeling with 2’-aminodeoxycytidine, structure-dependent 2’-amino reaction with an aromatic isothiocyanate, and high-throughput RNA sequencing. Our method maps cellular RNA structure transcriptome-wide with temporal resolution enabling determination of transcript age-dependent RNA structural dynamics. We benchmark our approach against known RNA structures and investigate the dynamics of human 5S rRNA during ribosome biogenesis, revealing specific structural changes in 5S rRNA loops that occur over the course of several hours. Taken together, our work sheds light on the maturation and coordinated conformational changes that take place during ribosome biogenesis and provides a general strategy for surveying evolving RNA structural dynamics across the transcriptome. SNIPER-Seq of six samples from HEK293T cells, including two treated with DMSO as control, two treated with 2’-aminodeoxycytidine and isoS-N3 in living cells, and two treated with2’-aminodeoxycytidine after RNA extraction and reacted with isoS-N3 in vitro