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 (m2G) and N 2,N 2-dimethylguanosine (m2 2G). Using visible light-mediated organic photoredox catalysis, m2G and m2 2G 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 m2 2G sites.