Signal Amplification of Graphene Oxide Combining with Restriction Endonuclease for Site-Specific Determination of DNA Methylation and Assay of Methyltransferase Activity

摘要

Site-specific identification of DNA methylation and assay of MTase activity are important in determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. This work reports an electrochemical method for gene-specific methylation detection and MTase activity assay using HpaII endonuclease to improve selectivity and employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The method was developed by designing a probe DNA, which was immobilized on electrode surface, to hybridize with target DNA (one 137 mer DNA from exon 8 promoter region of the Homo sapiens p53 gene, was extracted from HCT116 cells). The assay is based on the electrochemical responses of the reporter (thionine), which was conjugated to 3'-terminus of the probe DNA via GO, after the DNA hybrid was methylated (under catalysis of M.SssI MTase) and cleaved by HpaII endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5'-CCGG-3' and catalyzing cleavage between the cytosines). This model can determine DNA methylation at the site of CpG and has an ability to discriminate the target DNA sequence from even single-base mismatched sequence. The electrochemical signal has a linear relationship with M.SssI activities ranging from 0.1 to 450 U/mL with a detection limit of ～(0.05 ± 0.02) U/mL at a signaloise of 3. The advantages of this assay are ease of performance having a good specificity and selectivity. In addition, we also demonstrate the method can be used for rapid evaluation and screening of the inhibitors of MTase and has a potential application in discovery of new anticancer drugs.