A CpG methylation-powered dynamic three-dimensional-DNAzyme walker for single-molecule monitoring of multiple cytosine-C5 methyltransferases

Abstract

Cytosine-C5 methyltransferases (MTases) maintain methylation status in CpG islands (CpGIs), and their abnormal activities are implicated in various diseases and cancers. Simultaneous monitoring of multiple cytosine-C5 MTases has remained challenging due to diversity of DNA substrates and scarcity of methylation-independent restriction endonucleases (MIREs). Herein, we construct a CpG methylation-powered dynamic three-dimensional-DNAzyme walker for single-molecule monitoring of multiple cytosine-C5 MTases. The presence of M.SssI and M.CviPI catalyzes cytosines-C5 methylation in hairpin probes to form catalytic substrates of GlaI. Then GlaI specifically cleaves 5-mCs to yield two metal-specific DNAzymes. Upon the addition of substrate probes (SP1/SP2)-functionalized AuNPs, two DNAzymes selectively bind with SP1 and SP2, and autonomously complete 3D-DNAzymes walking with assistance of cofactors Mg2+ and Zn2+, inducing cyclic cleavage of substrate probes and dissociation of abundant Cy5 and Cy3 from AuNPs. Cy5 and Cy3 are simply measured through single-molecule imaging, with Cy5 indicating M.SssI and Cy3 indicating M.CviPI. This nanosensor achieves a limit of detection (LOD) of 3.944 × 10-9 U/μL for M.SssI and 5.046 × 10-9 U/μL for M.CviPI. It can discriminate cytosine-C5 MTases members, screen potential inhibitors, and quantify multiple cytosine-C5 MTases in human serum and diverse cell lysates, providing a powerful platform for biomedical research and clinical diagnostics.