Biosensor For DNA Methyltransferase Detection Based On DNA Hairpin And G-quadruplex And Application Research

DNA methylation, an essential part of epigenetics research, is an important link between genotype and phenotype. This significant biological activity is under the responsibility of methyltransferase(MTase), which catalyzes a transfer of a methyl group from S-adenosyl-L-methionine to adenine or cytosine in the target DNA. It is indicated that abnormal level of DNA MTase can cause aberrant DNA methylation which is closely associated with pathogenic mechanisms to human, especially cancer. Moreover, DNA MTase plays an important role in regulation of DNA methylation, which has great effect on chromatin structure adjustment and gene expression. As a result, various methods have been proposed for detection of DNA MTase in recent years. However, these methods have the disadvantages of expensive and complex. Therefore, simple, efficient and sensitive approaches for assay of DNA MTase activity are still a need.(1) We presented a new type of DNA functionalized nano mesoporous silica(MSNs) was creatively introduced to the detection of DNA MTase activity with Gquadruplex as a lock for signal molecule to release. The method was carried out by designing a particular DNA which could fold into G-quadruplex and complement with probe DNA. Next, MSNs was prepared before blocking methylene blue(MB) by Gquadruplex. Probe DNA was then fixed on gold nanoparticles modified glass carbon electrode, and the material was able to be transferred to the surface of electrode by DNA hybridization. After methylation of DNA MTase and the cutting of restriction endonuclease, the electrode was transferred to phosphate buffer solution(pH 9.0) for the releasing of MB. The response of differential pulse voltammetry was obtained from the release of MB. Consequently, the difference of signals with or without methylation could prove the assay of M. Sss I MTase activity. The results showed that the responses from MB increased linearly with the increasing of the M. Sss I MTase concentrations from 0.28 to 50 U/m L. The limit of detection was 0.28 U/mL. In addition, Zebularine, a nucleoside analog of cytidine, was utilized for studying the inhibition activity of M. Sss I MTase.(2) A simple electrochemical strategy for sensitive detection of DNA methyltransferase(MTase) and inhibitor screening based on DpnI digestion triggering the formation of G-quadruplex DNAzymes has been developed. In this paper, a probe richness of guanine(G) was first self-assembled on the surface of the electrode through Au-S bond and then hybridized with the complementary DNA. Without DNA methylation, G-quadruplex DNAzymes cannot be formed due to the double helix structure and a weak electrochemical response can be observed. On the contrary, an obvious enhancement of the electrochemical response can be achieved after the cleavage of the methylated double-strand DNA by DpnI since G-quadruplex DNAzymes can be obtained, which catalyze the oxidation of hydroquinone by H2O2 with the assistance of the cofactor hemin. This method is under a detection limit of 0.96 U/m L and can monitor the change of DNA methylation level selectively. Moreover, RG108 was selected as a representative inhibitor for studying the inhibition activity of DNA MTase.(3) We report a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine(polyA) DNA with a gold nanoparticles functioned glassy carbon electrode(AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue(MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27 U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery.