PhOxi-Seq: Single-Nucleotide Resolution Sequencing of N2-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 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.

Single-nucleotide resolution of N 6-adenine methylation sites in DNA and RNA by nitrite sequencing.

A single-nucleotide resolution sequencing method of N 6-adenine methylation sites in DNA and RNA is described. Using sodium nitrite under acidic conditions, chemoselective deamination of unmethylated adenines readily occurs, without competing deamination of N 6-adenine sites. The deamination of adenines results in the formation of hypoxanthine bases, which are read by polymerases and reverse transcriptases as guanine; the methylated adenine sites resist deamination and are read as adenine. The approach, when coupled with high-throughput DNA sequencing and mutational analysis, enables the identification of N 6-adenine sites in RNA and DNA within various sequence contexts.