Profiling demethylase activity using epigenetically inactivated DNAzyme

The level of DNA methylation has been reported to be closely associated with various carcinomas and is dynamically regulated by several demethylases. However, current demethylase detection methods are mainly antibody-based, which detect the demethylase concentrations, while the actual numbers of catalytically active demethylase remain unknown. Thus, we have developed an activity assay based on epigenetically modified DNAzymes (EMOzymes), and CRISPR/Cas12a facilitated cascade signal amplification. We have ultrasensitively quantitated the activity of O6-methylguanine-DNA-methyltransferase (MGMT), a key demethylase that contributes to the chemoresistance to alkylation agents in cancer therapy, with an estimated limit of detection of 0.054 nM. This approach opens a new avenue for sensitively profiling the activity of many disease-related demethylases.