Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N 6 -methyladenosine in RNA at one-nucleotide resolution.

N 6 -Methyladenosine (m6 A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m6 A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m6 A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m6 A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m6 A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m6 A can be determined and the selectivity is up to 54.1-fold to discriminate m6 A against unmodified adenosine (A). The sensitivity has been improved about 106 -fold so the proposed method can be successfully employed to accurately determine m6 A in real biological samples, even in low abundance RNA.