Nm-seq finds thousands of modified 2'-O-methylation sites in mRNA with base precision

Nm-seq maps 2''-O-methylation sites in human mRNA with base precision The ribose of rna nucleotides can be 2'-O-methylated (nm). despite advances in high-throughput detection, the inert chemical nature of nm still limits sensitivity and precludes mapping in mrna. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop nm-seq, a sensitive method for transcriptome-wide mapping of nm with base precision. nm-seq uncovered thousands of nm sites in human mrna with features suggesting functional roles. Overall design: we developed a conceptually distinct approach based on the different chemical properties of nucleosides with 2'-OH and 2'-OMe22–25, combining enrichment with detection of a positive signal (rather than the lack of signal) to produce a sensitive method suited for discovery of Nm sites in rare RNA molecules or at low stoichiometry. Nm-seq leverages oxidative cleavage of ribose 2',3'-vicinal diols by periodate to expose, enrich and map Nm sites in the transcriptome without bias and with single-nucleotide precision. Nm-seq first exposes internal Nm sites in RNA fragments by iterative oxidation–elimination–dephosphorylation (OED) cycles that remove 2'-unmodified nucleotides (one per cycle) in the 3'-to-5' direction.