Copper-Free Click Chemistry-Mediated Assembly of Single Quantum Dot Nanosensor for Accurately Monitoring Locus-Specific m⁶A in Cancer Cells

N6-methyladenosine (m(6)A) plays an important role in embryogenesis, nuclear export, transcription splicing, and protein translation control. Herein, we demonstrate a copper-free click chemistry-mediated assembly of single quantum dot (QD) nanosensor for accurately monitoring locus-specific m(6)A in cancer cells. The m(6)A-sensitive endoribonuclease MazF can digest the unmethylated A-RNA, and the intact m(6)A-RNA then hybridizes with DNA probes a and b to produce a sandwich hybrid, initiating the click chemistry to generate probe a-b ligation product via first tandem ligation detection reaction (LDR) cycle. Subsequently, DNA probes c and d can hybridize with the probe a-b ligation product to generate the probe c-d ligation product via second LDR cycle. Both LDR cycles can be repeated through denaturation and annealing reaction to generate abundant biotin-/fluorophore-modified probe c-d ligation products that can easily assemble on the QD surface to induce distinct fluorescence resonance energy transfer (FRET) between QD and Cy5. This assay can be homogenously performed without the involvement of copper catalyst, m(6)A-specific antibody, radioactive labeling, ligase enzyme, enzymatic reverse transcription, and next-generation sequencing. Moreover, it can discriminate even 0.01% m(6)A level in complex samples and accurately measure cellular m(6)A-RNA expression, providing a promising avenue for clinical diagnostics and biomedical research.