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.