The Transcriptome-Wide Mapping of 2-Methylthio-N6-isopentenyladenosine at Single-Base Resolution

Hundreds of modified bases have been identified and enzymatically modified to transfer RNAs (tRNAs) to regulate RNA function in various organisms. 2-Methylthio-N6-isopenteny-ladenosine (ms2i6A), a hypermodified base found at tRNA position 37, exists in both prokaryotes and eukaryotes. ms2i6A is traditionally identified by separating and digesting each tRNA from total RNA using RNA mass spectrometry. A transcriptome-wide and single-base resolution method that enables absolute mapping of ms2i6A along with analysis of its distribution in different RNAs is lacking. Here, through chemoselective methylthio group bioconjugation, we introduce a new approach (redox activated chemical tagging sequencing, ReACT-seq) to detect ms2i6A transcriptome-wide at single-base resolution. Using the chemoselectivity between the methylthio group and oxaziridine group, ms2i6A is bio-orthogonally tagged with an azide group without interference of canonical nucleotides, advancing enrichment of methylthio group modified RNAs prior to sequencing. ReACT-seq was demonstrated on nine known tRNAs and proved to be highly accurate, and the reverse transcription stop (RT-stop) character enables ReACT-seq detection at single-base resolution. In addition, ReACT-seq identified that the modification of ms2i6A is conservative and may not exist in other RNAs.

Automated summary

A new method called ReACT-seq is introduced to detect ms2i6A transcriptome-wide at single-base resolution, through chemoselective reactions between the methylthio group and oxaziridine group. This method demonstrates high accuracy and resolution, and reveals that the modification of ms2i6A is conservative and may not exist in other RNAs.