RNA solvent accessibility by hydroxyl radical probing. Escherichia coli strain:B strain

In recent years, different probing methods have been developed for deciphering RNA secondary and tertiary structural properties. The probe reactivity readout of these methods is based on either reverse transcription (RT) stop at the cleaving sites or mutational profiling at the adduct formation sites. These approaches are challenged with the existence of false-positive probing signals because of the early drop-off of reverse transcriptase, use of random primers in RT reaction, and PCR amplification bias. To overcome these pitfalls, we introduced a new approach named the high-throughput sequencing-based hydroxyl radical probed end targeted (HRPet-Seq) method. This technique uses hydroxyl radicals as a probing reagent and interrogates RNA structures by solvent accessibility study. A completely different way of detecting probing sites has been revealed in this method. Hydroxyl radicals cleave the solvent-exposed nucleotides and produce 3'-P ends containing fragments, which are identified by ligating a linker sequence with the help of RtcB ligase enzyme. The application of this method on E. coli ribosomes showed a strong correlation (SCC of 0.532, 0.500 and 0.518 for 16S, 23S and 5S rRNA, respectively) with the calculated solvent accessibility from high-resolution crystal structures. Interestingly, this method significantly avoided the PCR amplification bias and showed a strong agreement between the correlations with or without amplicons deduplication. The method excels at recovering accessibility signal at low sequencing depth by directly capturing relevant fragments by targeted RT-PCR. However, this HRPet-Seq is currently applicable to only in vitro studies, modifications and improvements are required for advancing it to in vivo as well as transcriptome-wide RNA structure probing.