Classical approaches to determine structures of noncoding RNA (ncRNA) probed only one RNA at a time with enzymes and chemicals, using gel electrophoresis to identify reactive positions. To accelerate RNA structure inference, the authors developed fragmentation sequencing (FragSeq), a high-throughput RNA structure probing method that uses high-throughput RNA sequencing of fragments generated by digestion with nuclease P1, which specifically cleaves single-stranded nucleic acids.
In experiments probing the entire mouse nuclear transcriptome, they accurately and simultaneously mapped single-stranded RNA regions in multiple ncRNAs with known structure. They probed in two cell types to verify reproducibility and also identified and experimentally validated structured regions in ncRNAs with, to our knowledge, no previously reported probing data.
Underwood JG, Uzilov AV, Katzman S, Onodera CS, Mainzer JE, Mathews DH, Lowe TM, Salama SR, Haussler D. (2010) FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat Methods [Epub ahead of print]. [abstract]