Sequential structure probing of cotranscriptional RNA folding intermediates.

Cotranscriptional RNA folding pathways typically involve the sequential formation of folding intermediates. Existing methods for cotranscriptional RNA structure probing map the structure of nascent RNA in the context of a terminally arrested transcription elongation complex. Consequently, the rearrangement of RNA structures as nucleotides are added to the transcript can be inferred but is not assessed directly. To address this limitation, we have developed linked-multipoint Transcription Elongation Complex RNA structure probing (TECprobe-LM), which assesses the cotranscriptional rearrangement of RNA structures by sequentially positioning RNAP at two or more points within a DNA template so that nascent RNA can be chemically probed. We validated TECprobe-LM by measuring known folding events that occur within the signal recognition particle RNA, ZTP riboswitch, and fluoride riboswitch folding pathways. Our findings establish TECprobe-LM as a strategy for detecting cotranscriptional RNA folding events directly using chemical probing.