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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.

chemical science

Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of <i>N</i><sup>6</sup>-methyladenosine in RNA at one-nucleotide resolution

Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N6-methyladenosine in RNA at one-nucleotide resolution

Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N⁶-methyladenosine in RNA at one-nucleotide resolution