Development of Mild Chemical Catalysis Conditions for m 1 A-to-m 6 A Rearrangement on RNA.

The conversion of N 1 -methyladenosine (m 1 A) to N 6 -methyladenosine (m 6 A) on RNA is an important step for both allowing efficient reverse transcription read-though for sequencing analysis and mapping modifications in the transcriptome. Enzymatic transformation is often used, but the efficiency of the removal can depend on local sequence context. Chemical conversion through the application of the Dimroth rearrangement, in which m 1 A rearranges into m 6 A under heat and alkaline conditions, is an alternative, but the required alkaline conditions result in significant RNA degradation by hydrolysis of the phosphodiester backbone. Here, we report novel, mild pH conditions that catalyze m 1 A-to-m 6 A arrangement using 4-nitrothiophenol as a catalyst. We demonstrate the efficient rearrangement in mononucleosides, synthetic RNA oligonucleotides, and RNAs isolated from human cell lines, thereby validating a new approach for converting m 1 A-to-m 6 A in RNA samples for sequencing analyses.