Affiliation:
1. Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Henan Institute of Medical and Pharmaceutical Sciences BGI College, Zhengzhou University Zhengzhou Henan 450052 China
2. College of Chemistry, Chemical Engineering and Materials Science Shandong Normal University Jinan Shandong 250014 China
3. School of Chemistry and Chemical Engineering Southeast University Nanjing Jiangsu 211189 China
Abstract
Comprehensive SummaryN6‐methyladenosine (m6A) 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 m6A in cancer cells. The m6A‐sensitive endoribonuclease MazF can digest the unmethylated A‐RNA, and the intact m6A‐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, m6A‐specific antibody, radioactive labeling, ligase enzyme, enzymatic reverse transcription, and next‐generation sequencing. Moreover, it can discriminate even 0.01% m6A level in complex samples and accurately measure cellular m6A‐RNA expression, providing a promising avenue for clinical diagnostics and biomedical research.