Construction of a N6-methyladenosine demethylation-switched deoxyribozyme circuit for in vitro and in situ detection of FTO activity

Fat mass and obesity-associated proteins (FTO) can erase the methyl modification on N6-methyladenosine (m6A), and the aberrant expression level of FTO is tightly linked to diverse human diseases (e.g., cancers, Alzheimer's diseases, diabetes, obesity, and acute myeloid leukemia). Herein, we construct a m6A demethylation-switched deoxyribozyme (DNAzyme) circuit for in vitro and in situ detection of FTO activity. In this assay, a m6A-caged DNAzyme substrate probe is engineered as a switch of the sensor. The presence of target FTO can catalyze demethylation reaction to restore the DNAzyme catalytic activity. Subsequently, the active DNAzymes cyclically cleave hairpin probes 1 (HP1) to liberate abundant triggers. The resulting triggers can catalyze unceasing hybridization between two hairpin probes (i.e., Cy3-labeled HP2 and Cy5-labeled HP3) through toehold-mediated strand displacement (TMSD) reaction, generating long fluorescent dsDNA nanowires and simultaneously bringing two fluorophores (e.g., Cy3 and Cy5 molecules) into close proximity, in which efficient Förster resonance energy transfer (FRET) occurs from Cy3 molecular to Cy5 molecular. This sensor displays excellent specificity, good stability and high sensitivity with a detection limit down to 9.47 × 10−15 M (9.47 fM), and it can be used for screening of FTO inhibitor, kinetic analysis, and monitoring of dynamic expression of endogenous FTO in living cells. Moreover, this nanosensor can discriminate the FTO expression level in breast cancer patient tissues from healthy individual tissues, providing a potential platform for m6A demethylase-related biological study, drug discovery, and clinical diagnosis.