Chemical manipulation of m 1 A mediates its detection in human tRNA.

N 1 -methyl adenosine (m1 A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1 A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1 A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1 A. In this study, we introduce a reduction-based m1 A sequencing (red-m1 A-seq). We report that NaBH4 reduction of m1 A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1 A to m1 A to provide good controls, allowing the detection of m1 A with higher sensitivity and accuracy. We applied red m1 A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1 A sites, but also new m1 A sites in mt-tRNAAsn-ATT and 5.8S rRNA.