PhOxi-Seq: Single-Nucleotide Resolution Sequencing of N2-Methylation at Guanosine in RNA by Photoredox Catalysis

Abstract

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 N²-methylation, a common methylation mark seen in N²-methylguanosine (m²G) and N²,N²-dimethylguanosine (m²₂G). Using visible light-mediated organic photoredox catalysis, m²G and m²₂G 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 N²-methylation with excellent response and markedly improved read-through at m²₂G sites.