Construction of Multiple DNAzymes Driven by Single Base Elongation and Ligation for Single-Molecule Monitoring of FTO in Cancer Tissues

Fat mass and obesity-associated proteins (FTO) play anessentialrole in the reversible regulation of N-6-methyladenosine(m(6)A) epigenetic modification, and the overexpression ofFTO is closely associated with the occurrence of diverse human diseases(e.g., obesity and cancers). Herein, we demonstrate the constructionof multiple DNAzymes driven by single base elongation and ligationfor the single-molecule monitoring of FTO in cancer tissues. Whentarget FTO is present, the m(6)A-RNA is specifically demethylatedand subsequently acts as a primer to combine with the padlock probe,initiating single-base elongation and ligation reaction to generatea closed template probe. Upon the addition of phi29 DNA polymerase,a rolling circle amplification (RCA) reaction is initiated to producelarge numbers of Mg2+-dependent DNAzyme repeats. Subsequently,the DNAzymes cyclically digest the signal probes, liberating numerousCy5 molecules that can be precisely counted by single-molecule imaging.Taking advantage of the sequence specificity of the polymerase/ligase-mediatedgap-filling and ligation as well as the high amplification efficiencyof RCA, this biosensor shows excellent specificity and high sensitivitywith a detection limit of 5.96 x 10(-16) M. Itcan be applied to screen FTO inhibitors and quantify FTO activityat the single-cell level. Moreover, the proposed strategy can accuratelydistinguish the FTO expression level in tissues of healthy individualsand breast cancer patients, providing a new platform for drug discovery,m(6)A modification-related research, and clinical diagnostics.