Electrochemical Detection Of DNA Methyltransferase Based On Gold Nanoparticles And Hairpin DNA Probe

DNA methylation, a critical life process existing in both prokaryotes andeukaryotes, is achieved through the catalysis of DNA methyltransferases （MTases） totransfer a methyl group from the cofactor S-adenosyl-L-methionine （SAM） to thetarget cytosine or adenine in the specific recognized DNA sequences. As a commonnucleic acid modification, DNA methylation is involved in chromatin structure, DNAconformation, DNA stability and the interaction between DNA and protein. AberrantDNA methylation can change normal cellular functions and phenotypes, which mayresult in tumor occurrence and tumor growth. Therefore, the detection of MTasesactivity and analysis of DNA methylation process are crucial to life science researchand early cancer diagnosis. However, conventional methods for quantifying DNAMTase and analyzing DNA methylation are usually time-intensive, DNA-consuming,laborious, or require isotope labeling, thus leading to an urgent need for novel DNAMTase and DNA methylation assay methods. Electrochemical biosensors, possessingseveral advantages such as high sensitivity, simple operation, fast response, easy ofminiaturization and cost-effectiveness, have been widely explored for the detection ofvarious targets. In this paper, utilizing the nanomaterial-based signal amplificationand the smart design of hairpin-structured DNA probes, we constructed a series ofelectrochemical sensors to sensitively detect the MTase activity. The main works aresummarized as follows:1. A sensitive signal-on electrochemical assay for MTase activity using AuNPsamplificationA sensitive and simple signal-on electrochemical assay for detection of Dammethyltransferase （MTase） activity based on DNA-functionalized gold nanoparticals（AuNPs） amplification coupled with enzyme-linkage reactions is presented. This newassay takes advantage of the steric hindrance of AuNPs and the electrostatic repulsionbetween the negative-charge phosphate backbones of DNA modified on the AuNPsand redox probe [Fe（CN）₆]^3-/4-. In this method, the self-assembled ssDNA on theelectrode is hybridized with its complement ssDNA modified on AuNPs to formdsDNA AuNPs bioconjugates containing specific recognition sequence of DamMTase and methylation-sensitive restriction endonuclease Dpn I. Then, the AuNPsapproach to the electrode and result in blockage of electronic transmission. It is eT OFF state. In the presence of Dam MTase and Dpn I, the specific sequence ismethylated and cleavaged, which in turn release the DNA modified AuNPs from theelectrode surface allowing free exchange of electrons. It generates a measurableelectrochemical signal （eT ON）. Under optimized conditions, a detection limit of0.12U/mL are obtained. Furthermore, our new assay is a promising method to screeninhibitors or drugs for Dam MTase.2. A sensitive signal-on electrochemical assay for MTase activity based onmethylation-responsive hairpin-capture DNA probeThis work develops a simple, sensitive and signal-on electrochemical sensor formethyltransferase （MTase） activity analysis. The sensor is composed of a methyleneblue-modified "signaling DNA probe" and a "capture DNA probe" tetheredmethylation-responsive hairpin DNA （hairpin-capture DNA probe）. The thiol-modified hairpin-capture DNA probe at5′end was firstly self-assembled on goldelectrode via Au–S bonding. Methylation-induced scission of hairpin-capture DNAprobe would displace the hairpin section and remain the "capture DNA probe"section on the gold electrode. Subsequently, the remained "capture DNA probe" onthe gold electrode can hybridize with the methylene blue-modified "signaling DNAprobe", mediating methylene blue onto the gold electrode surface to generate redoxcurrent. It was eT on state. The developed facile signal-on electrochemical sensingsystem showed a linear response to concentration of Dam MTase range from0.1to1.0U/mL. The detection limit of Dam MTase activity was determined to be0.07U/mL and the total detection time is7h. The sensor also has the ability to provideinformation about the dynamics of methylation process. Furthermore, wedemonstrated that this sensor could be utilized to screen inhibitors or drugs for DamMTase.3. A sensitive signal-on electrochemical assay for MTase activity based onmethylation-responsive G-quadruplex-incorporated DNA probeAn electrochemical method for detection of MTase activity was developed basedon a methylation-responsive G-quadruplex-incorporated hairpin DNA probe. Thestem of the hairpin DNA probe was designed to incorporate the specific recognitionsequence of Dam MTase and a G-rich region. Via Au–S bonding, the thiol-modifiedmethylation-responsive G-quadruplex-incorporated hairpin DNA probe was firstlyself-assembled on gold electrode. Methylation-induced scission of the hairpin DNAprobe would activate the binding of the G-quadruplex with Hemin, which was used asthe electroactive probe. This resulted in the measurable electrochemical signal obtained, which could accurately reflect the activity of Dam MTase indirectly. Thismethod is fast, simple and sensitive with a detection limit of0.2U/mL.