Assembly of Dandelion-Like Nanoprobe for Sensitive\nDetection of N6-Methyladenosine Demethylase by Single-Molecule Counting

N6-methyladenosine\n(m6A) demethylase is essential for enzymatically\nremoving methyl groups from m6A modifications and is significantly\nimplicated in the pathogenesis and advancement of various cancers,\nwhich makes it a promising biomarker for cancer detection and research.\nAs a proof of concept, we select the fat mass and obesity-associated\nprotein (FTO) as the target m6A demethylase and develop a dandelion-like\nnanoprobe-based sensing platform by employing biobar-code amplification\n(BCA) for signal amplification. We construct two meticulously designed\nthree-dimensional structures: reporter-loaded gold nanoparticles (Reporter@Au\nNPs) and substrate-loaded magnetic microparticles (Substrate@MMPs),\nwhich can self-assemble to form dandelion-like nanoprobes via complementary\nbase pairing. In the presence of FTO, the m6A-containing substrates\nare demethylated, triggering the MazF-assisted cleavage reaction and\nthereby releasing the Reporter@Au NPs. Furthermore, upon digestion\nby exonucleases, the Reporter@Au NPs may liberate a significant quantity\nof Cy3 signals. Remarkably, the combined effects of Au NPs’\nsuperior enrichment capacity, MMPs’ exceptional magnetic separation\nefficiency, and the precision of the single-molecule detection platform\nendow the FTO sensor with exceptional sensitivity and specificity\nwith a detection limit of 7.46 × 10<sup>–16</sup> M. Additionally,\nthis method offers a versatile platform for the detection of m6A demethylase\nand the screening of corresponding inhibitors, thereby advancing clinical\ndiagnosis and drug development.