Electrochemical Analysis Of DNA Methyltransferase Activity And Hydroxymethylation Degree Based On DNA Hybridization Chain Amplification Strategy

DNA methylation is one of the hottest research areas on epigenetic modification of genomic DNA. DNA methylation catalyzed by DNA methyltransferase (MTase), During early development, the mammalian DNA methylation is stable epigenetic states, but many researches have reported that DNA methylation may be reversible in mammalian cells, which is known as active demethylation process. But the demethylation process is more complicated with respect to the process of methylation. The two processes of DNA methylation and demethylation are balanced with each other, maintaining the stability of DNA methylation patterns. According to numerous studies, DNA methylation plays a critical role in embryonic development and cell cycle regulation as well as the pathogenesis of various human diseases such as cancers. Therefore, it is essential to develop simple, sensitive, accurate, and reliable approach for locating and quantitative detection of DNA methylation. The elechemical techniques have been considered to be promising for determinations because of the advantages such as high sensitivity, low cost, easy miniaturization and automation. The aim of thesis is to develop a electrochemical approach used for determination of DNA methylayion level, analysis the activity of methyltransferase, evaluation and screen of the inhibitors of methytrasferase and quantification of hydroxymethylation of DNA demethylation. As a general method of DNA detection, these studies are expected to be applicable to type of DNA analysis. It has high potential application in molecular diagnostics of disease in clinical environment. The aim results are as follows:1. The DNA methyltransferase (MTase) activity is highly correlated with the occurrence and development of cancer. This work reports a label-free superstructure-based electrochemical assay for signal-amplified DNA MTase (using M.SssI MTase as an example) activity detection. The controlled low density of immobilized DNA is essential in the information of DNA superstructure which can help the efficient signal amplification. By using RuHex that are binded to the DNA double strands as electroactive reporter, differential pulse voltammetry (DPV) could quantitatively detection the activity of DNA MTase within the range of 0.5 to 120 U/mL with a detection limit of 0.025 U/mL. In addition, we also demonstrated the application of the developed method for a rapid evaluation and screening of the inhibitors of MTase, which may be a help for the discovery of methylation-related anticancer drugs.2. DNA demethylation process is a reversible process of DNA methylation. But it is not successful for directly remove the methyl group of the methylated cytosines for many years. The effective studies have identified that cytosines in mammalian cells can be oxidized to 5hmC (5-hydroxymethylcytosine) by the Tet family of enzymes. And these findings suggest that 5hmC provides a new idea for the study of DNA demethylation.5hmC has important roles in somatic cell reprogramming, embryonic development, and initiating the DNA demethylation in mammalian. Therefor, it is essential to develop new methods and technologies for detecting and sequencing 5hmC. In this work, we develop a label-free, superstructure-based electrochemical method to effective quantify DNA demethylation. Once the hydroxymethylated CpG site in the 5’-CCGG-3’is in the presence of T4-βGT, MspJI recognition is blocked. Then, FcA-moiety can promote the electron transfer between gold electrodes and redox-labeled DNA duplexes in the presence of T4-βGT and helper DNA when Ru(NH3)63+molecules were electrostatically attached to the DNA duplex of the hydroxymethylated dsDNA biosensor. (I2-I0/I1-I0) is represents the hydroxymethylated proportion of ds-DNA. In this paper, an obvious increase in (I2-I0/I1-I0) was achieved in the hydroxymethylation degree ranges from 1%to 90% in a linear relation with the detection limit estimated to be 0.05% at a signal/noise of 3 Therefor, this approach can amplify the measured electrochemical signal and thus enhance the assay sensitivity.