DNAzyme mediated catalytic hairpin self-assembly strategy enables the sensitive and facile investigation of N-methyladenine

N6-methyladenosine (m6A) is a crucial post-transcriptional epigenetic modification in RNAs which associates with numerous cancers development. Here, a cost-effective, and easy-to-operate fluorescence strategy for m6A analysis was developed based on the different cleavage efficiency of 8-17 DNAzyme for methylation and unmethylation sites. By arranging 8-17 DNAzyme to recognize and cleave 5'-rAG-3' and 5'-rm6AG-3' motifs in the target sequence respectively, various short ssDNA oligoes were produced, leaving different amounts of target sequence. Benefiting from the varying degrees of retardation of CHA amplification reaction, the tiny discrepancy between m6A and unmethylated adenine (A) can be distinguished. Moreover, we found that 8-17 DNAzyme was more sensitive to m6A than to A. Importantly, the strategy we proposed not only avoids sophisticated chemical labelling and expensive proteins, but also can be easily performed on the ordinary heating apparatus of accessible laboratory. The DCHA strategy demonstrated good analytical performance in complex biological matrices, and was successfully applied to hepatocellular carcinoma-related m6A analysis in CTNNB1, MGAT5 and SOCS-2 mRNAs with satisfactory results, showing its potential in clinical analysis.