PhOxi-Seq: Single-Nucleotide Resolution Sequencing of <i>N</i> <sup>2</sup> -Methylation at Guanosine in RNA by Photoredox Catalysis
Kimberley Chung Kim Chung, Yasaman Mahdavi‐Amiri, Christopher Korfmann, Ryan Hili
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.