Demethylase FTO activity analysis based on methyl sensitive enzyme MazF and hybridization chain reaction

Abstract

FTO mediates oxidation demethylation of different RNA types and regulates fat mass and adipogenesis. More and more evidences show that FTO is deeply involved in the development of human diseases, such as acute myeloid leukemia and lung cancer, by regulating the content of m6A. Hence, it is essential to detect FTO in a smart way. At present study, we have developed a simple, sensitive and reliable methyl-sensitive nucleotide biosensor to analyze the activity of demethylase based on the methyl-sensitive RNA restriction enzyme MazF and the hybridization chain reaction (HCR) as a signal amplification unit. In the presence of target FTO, the biotin-modified 3′end of m6A RNA probe was demethylated and became available for MazF digestion, afterwards the trigger sequence obtained by digestion could continue to open hairpin structure for HCR. Under the optimal conditions, the linear range of this method was 20 nM to 200 nM with the detection limit of 2.273 nM. This strategy provided outgoing sensitivity, specificity and anti-interference ability in MDA-MB-231 cell lysates. In addition, we verified the feasibility of this strategy for FTO inhibitors and drug screening. Furthermore, this strategy could be used for analyzing other demethylases of m6A.