New Approach For Methyltransferase Activity And Biothiols Assay

DNA methylation plays an important role in the regulation of gene epigenetic modification in the human genome. During this aberrant methylation process, the DNA MTase acts as a crucial participator to transfer a methyl group from S-adenosylmethionine(SAM) to the C5-position of cytosine in CpG islands. Recent research demonstrates that abnormal methylation of CpG islands in the promoter region of the gene is a new generation of cancer biomarkers. However, traditional methods of methylase activity analysis were time consuming and operating complex. Thus, a sensitive approach for the rapid detection of DNA methylation and the assay of MTase activity could provide a powerful method for early cancer diagnosis. The main results of this research are given as follows:(1) Ultrasensitive Electrochemical Detection of DNA Methyltransferase Based on Autocatalytic and Exonuclease III-Assisted Recycling Amplification Strategy. First,the Fc modified on the duplex DNA probe(S1/S2) could get closer to the electrode surface, leading to the increase of its current. In the presence of DNA methylation MTase(Dam), the hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then releasing of S3 sequence. The released S3 sequence hybridizes with the dangling ssDNA at the 3′ end of duplex DNA probe modified on Au electrode. Then the S2 sequence in the duplex DNA probe could be stepwise removed by Exo III accompanied that the releasing of Fc from the sensing interface and autonomous generation of new secondary S3 for the successive hybridization and cleavage process. Simultaneously, numerous S1 sequences on the electrode surface fold into G-quadruplex-hemin complexes with the help of K+ and hemin on the electrode surface to give a remarkable electrochemical response. Such conformational changes result in the decreasing of the DPV peak current of Fc and the increasing of the DPV peak current of G-quadruplex-hemin complexes, and the changes of dual signals are linear with the concentration of DNA methyltransferase.Because of this autocatalytic target recycling ampli?cation. The sensor enabled the assay of MTase activity with an impressive detection limit as low as 4.8×10-3 U/m L, much lower than those of most existing methods. Owing to the speci?c site recognition of MTases, this new approach could discriminate Dam MTase from other MTases such as M.SssI with high selectivity. Furthermore, the inhibition effects of several antibiotics are also investigated; indicating mitomycin and platinol have almost no effect on the methylation. However, benzylpenicillin and 5-?uorouracil can inhibit the methylase. The inhibition ef?ciency of 5-?uorouracil is the most serious one.(2) Label-Free Colorimetric Detection of Biothiols Utilizing SAM and Unmodified Au Nanoparticles. SAM contains a high-energy chiral sulfonium ion with methyl, aminopro-pylcarboxylate, and 5′-deoxyadenosine substituents bonded to the central sulfur atom. It should be mentioned that the sulfonium of the SAM molecule is positively charged because the extranuclear electron of sulfonium would transfer to the oxygen of 5′-deoxyadenosine with high electronegativity. SAM can induce aggregation of AuNPs due to SAM shelters the surface charge on the Au NPs, and the electrostatic repulsion between adjacent AuNPs decreases and the color changes from red to blue. In the presence of biothiols would prohibit the aggregation of AuNPs by SAM through formation of stable Au-S bonds between biothiols and AuNPs, which renders the biothiols molecule to encapsulate AuNPs in priority, leaving little or no chance for SAM binding on the AuNPs. Thus the biothiols can effectively cause the anti-aggregation of AuNPs, preserves the AuNPs as a red color. The corresponding color variation in the process of anti-aggregation of AuNPs can be used for the quantitative screening of biothiols through UV-vis spectroscopy or by the naked eye. Under optimized conditions, the range of 0.4-1.2 μM is obtained for Cys, 0.2-0.9 μM for GSH, and 0.6-3.0 μM for Hcys. The detection limits of this assay for GSH, Cys and Hcys are 35.8 nM, 21.7 nM, and 62.4 nM, respectively. The detection sensitivity of the proposed sensing system was found to be higher or comparable than those of other colorimetric methods. The determination of total biothiol content in human serum was performed. The recovery results ranged from 96.5% to 104.4%, indicating that no signi?cant interference with the determination of biothiols in serum samples.