Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/μL, which is superior to conventional ELISA (36.3 pg/μL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.