PhOxi-Seq: Single-Nucleotide Resolution Sequencing ofN2-Methylation at Guanosine in RNA by Photoredox Catalysis

Chemical modifications regulate the fate and function of cellular RNAs. Newly developed sequencing methods haveallowed a deeper understanding of the biological role of RNA modifications; however, the vast majority of post-transcriptionalmodifications lack a well-defined sequencing method. Here, we report a photo-oxidative sequencing (PhOxi-seq) approach forguanosineN2-methylation, a common methylation mark seen inN2-methylguanosine (m2G) andN2,N2-dimethylguanosine (m22G).Using visible light-mediated organic photoredox catalysis, m2G and m22G are chemoselectively oxidized in the presence of canonicalRNA nucleosides, which results in a strong mutation signature observed during sequencing. PhOxi-seq was demonstrated on varioustRNAs and rRNA to revealN2-methylation with excellent response and markedly improved read-through at m22G sites.