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 (m(6)A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m(6)A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m(6)A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m(6)A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m(6)A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m(6)A can be determined and the selectivity is up to 54.1-fold to discriminate m(6)A against unmodified adenosine (A). The sensitivity has been improved about 10(6)-fold so the proposed method can be successfully employed to accurately determine m(6)A in real biological samples, even in low abundance RNA.