Antibody-free enzyme-assisted chemical approach for detection of N(6)-methyladenosine.

The inert chemical property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunoprecipitation and cannot distinguish m6A and N6,2′-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, we present an antibody-free, FTO-assisted chemical labeling method termed m6A-SEAL for specific m6A detection. We applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, we demonstrated that m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO’s oxidation property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications. An antibody-independent m6A profiling method called m6A-SEAL was developed via turning m6A into stable dm6A after treatment by FTO and DTT. This method exhibits better reliability in detection of transcriptome-wide m6A sites in humans and plants.