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

Abstract

<i>N</i>⁶-Methyladenosine (m⁶A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m⁶A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m⁶A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m⁶A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m⁶A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m⁶A can be determined and the selectivity is up to 54.1-fold to discriminate m⁶A against unmodified adenosine (A). The sensitivity has been improved about 10⁶-fold so the proposed method can be successfully employed to accurately determine m⁶A in real biological samples, even in low abundance RNA.