PhOxi-Seq: Single-Nucleotide Resolution Sequencing of N 2 -Methylation at Guanosine in RNA by Photoredox Catalysis.

Chemical modifications regulate the fate and function of cellular RNAs. Newly developed sequencing methods have allowed a deeper understanding of the biological role of RNA modifications; however, the vast majority of post-transcriptional modifications lack a well-defined sequencing method. Here, we report a photo-oxidative sequencing (PhOxi-seq) approach for guanosine N 2 -methylation, a common methylation mark seen in N 2 -methylguanosine (m 2 G) and N 2 , N 2 -dimethylguanosine (m 2 2 G). Using visible light-mediated organic photoredox catalysis, m 2 G and m 2 2 G are chemoselectively oxidized in the presence of canonical RNA nucleosides, which results in a strong mutation signature observed during sequencing. PhOxi-seq was demonstrated on various tRNAs and rRNA to reveal N 2 -methylation with excellent response and markedly improved read-through at m 2 2 G sites.