New Approach Of Colorimetric Sensor Based On Strand Displacement Amplification For Methyltransferase Activity

DNA methylation is one of the most important epigenetics event and has been one of the most adequately studied epigenetics event. The process of DNA methylation will significantly influence the expression of genes. Alterations of methyltransferases activity may lead to aberrant DNA methylation patterns that are associated with several genetic diseases and various types of cancer. Thus, sensitive activity assay and inhibitor(anti-methylation drugs) screening for MTases represent a valuable strategy to both clinical diagnostics and therapeutics. This thesis is concentrated on study the activity of methyltransferases and design colorimetric sensor based on AuNPs. The main contents of this thesis are as follows:1. This section introduced DNA methylation and colorimetric sensor based on AuNPs. DNA methylation, a common gene protection approach, plays an important role in both prokaryotes and eukaryotes. It is extremely important to lots of normal cellular processes including development, transcriptional silencing, gene regulation, and X chromosome inactivation, among others. At present, the main approaches to study DNA methylation are analysis the activity of methyltransferases and the corresponding inhibitors. With the advantages of AuNPs, such as biological compatibility, easy to modifyed the surface, color changed obviously after aggregation, the colorimetric sensor based on AuNPs are widely used in biological detection.2. For the past few years, electrochemical sensor is the most common used sensor for methyltransferases assay. In order to enrich the research methods, this work developed colorimetric sensor for M.Sss I activity. We adopted the well designed HPI as methylated substrate and the ssDNA modified on AuNPs acted as probe DNA. The aggregation of AuNPs was based on the hybridization of DNA. Our method response well to the concentration of M.SssI in the range from 2 to 50 U/mL with the detection limit of 0.84 U/mL. The proposed method is simple with high selectivity.3. We present a colorimetric method for the assay of DNA methyltransferase(MTase) activity based on strand displacement amplification(SDA). In our design, hairpin DNA I(HPI) containing the sequence of 5’-CCGG-3’, which is specifically recognized by Cp G methyltransferase(M.Sss I) and Hpa II endonuclease. The methylated HPI would coexist with all the DNA and enzymes in the solution. While the unmethylated HPI can be cleaved into single-stranded DNA(ssDNA) fragments. The amplification is triggered by region I hybridization with another hairpin structure DNA II(HPII) to form a duplex, and then region I is replaced by probe DNA, thus lead to region I released from the duplex and triggered the cycle anew. Thus, resulted in the aggregation of AuNPs, and a concomitant color change from red to pale. We got a linear correlation of A/A0 vs logCM.Sss I when M.Sss I concentration ranging from 0.2 to 50 U/mL. The detection limit is calculated to be 0.08 U/mL. The sensitivity of strand displacement amplification(SDA) assay is higer than that direct assay we designed. In addition, the developed assay can also be applied to screen the inhibitors of M.SssI. The IC50 of 5-Aza and 5-Aza-dC are 2.4 and 0.37 μM respectively.