Chemiluminescence resonance energy transfer biosensing platform for site-specific determination of DNA methylation and assay of DNA methyltransferase activity using exonuclease III-assisted target recycling amplification

摘要

Site-specific determination of DNA methylation and assay of MTase activity can be used for determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. Herein, we develop a simple and highly sensitive chemiluminescence (CL) biosensing platform for site-specific determination of DNA methylation using Exonuclease III (Exo III)-assisted target recycling signal amplification. After bisulfite treatment of mixture of methylated DNA and unmethylated DNA, methylated DNA can hybridize with fluorescein (FAM)- labeled probe DNA to form double-stranded DNA (dsDNA), removing the FAM-labeled probe DNA from the surface of grapheme oxide, and the chemiluminescence resonance energy transfer (CRET) sensing signal can be observed and then amplified using Exo III-based recycling strategy. The biosensing platform exhibits excellent high sensitivity, and it can ever distinguish as low as 0.002% methylation level from the mixture, which is superior to most currently reported methods used for DNA methylation assay. In addition, the proposed method can also be used to sensitively assay MTase activity with determination limit of 0.007 U/mL. This CL biosensing offers the advantages of being facile, sensitive, rapid and cost-effective. These features make the system promising for future use for early cancer diagnosis and discover of new anticancer drugs.