Detection Of DNA Methylation Based On Thiazole Orange And Polyaniline

DNA methylation is an important epigenetic modification approach, which has frequently occurred at the carbon 5 position of C in CpG islands (5’-CG-3’) under the calalysis of methyltransferase. DNA methylation plays an important role in the regulation of gene transcription, eukaryote development, and cellular processes as well as the pathogenesis of various human diseases such as cancers. In normal cells, most CpG islands spanning the promoter regions are unmethylated, so their downstream genes are active. However, the CpG islands in cancer cells are methylated, and their downstream genes are silenced. Abnormal methylation in CpG islands can result in the change of normal cellular functions and phenotypes, and has been considered as a potential biomarker of diseases. Moreover, analyzing the MTase activity is also important in the regions of clinical diagnostics because DNA MTase catalyzes the covalent addition of the methyl group to C in DNA in the formation of mC. Therefore, a fast, cheap, simple, and selective method for detection of DNA methylation and M.SssⅠ MTase activity is imperative. In this paper, two works about the DNA methylation and DNA methyltransferase are as follows:Strategies to detect the methylation of site specific DNA and assay of M.SssⅠ methyltransferase (M.SssⅠ MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaⅡ and exonuclease Ⅲ (Exo Ⅲ). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaⅡ and degradation reaction of Exo Ⅲ, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.Sss Ⅰ MTase, the action of Hpa Ⅱ and Exo Ⅲ was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.In this work, we also proposed a novel electrochemical strategy for sensitive detection of DNA methylation and MTase activity based on polyaniline (PANT) as a redox mediator which was guided and deposited by HRP mimicking DNAzyme as a powerful catalyst and temp late of dsDNA. The biosensor was fabricated with the capture DNA probe (S1) first immobilized on Au electrode and hybridized with the target DNA (S2) to form double stranded DNA (dsDNA). In the absence of M.SssI MTase, the dsDNA was cleaved by restriction endonuclease HpaII. Upon the cleavage action of HpaII, DNAzyme DNA (S3) could not hybridize with S1, HRP-mimicking DNAzyme could not form and PANI could not deposit on the surface of Au electrode, leading to a low current signal. However, in the presence of M.SssI MTase, the S1/S2 hybrid was methylated and not cleaved by HpaII. Then, HRP-mimicking DNAzyme was successfully formed, which subsequently catalyzed the oxidation of aniline to PANI and resulted in a high current signal. This method can determine DNA methylation at the site of CpG. The human serum samples had been demonstrated. In addition, we also study the suitability of this biosensor for screening MTase inhibitor and this approach has a potential application in sensitive assay of a variety DNA MTases and screening potential drugs.