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

Abstract

N⁶-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.

Fig. 1. The general outline of the ligation-dependent PCR assay for m⁶A detection.

Fig. 2. Gel electrophoresis for identifying a ligase with substantial selectivity towards m⁶A. The probes used in the reactions were probe R1 D (a) and probe R1 (b).

Fig. 3. Gel electrophoresis for assessing the effects of core sequences on specificity.

Fig. 4. Specificity and sensitivity of the ligase-dependent PCR assay: (a) real-time fluorescence curves produced by the RNA2577-A segment in the concentration range from 4 fM to 4 nM under optimum experimental conditions. (b) The relationship between C_T and lg of RNA2577-A concentration (M). (c) Real-time fluorescence curves produced by the RNA2577-m⁶A segment in the concentration range from 4 fM to 4 nM under optimum experimental conditions.

Fig. 5. Determination of m⁶A in poly A⁺ RNA of HeLa cells. (a) Real-time fluorescence curves produced by the RNA2488-A segment in the concentration range from 4 fM to 4 nM. (b) The linear relationship between C_T and lg of RNA2488-A concentration (M). (c) Real-time fluorescence curves produced by RNA2488-A in 85 ng poly A⁺ RNA of HeLa cells under optimum experimental conditions. (d) Real-time fluorescence curves produced by RNA2577-A in 85 ng poly A⁺ RNA of HeLa cells under optimum experimental conditions.