New Biosensors For The Detection Of DNA MTase Activity

Epigenetics plays a significant role in maintaining specific cell phenotypes and creating biodiversity,and it is also essential to stabilize the structure of DNA and chromatin.DNA methylation,one of the most important and common epigenetic modification in mammal genomes,is the process of covalent modification of nucleotides in DNA with methyl groups.According to the type of nucleotides and site of methylaiton,DNA methylation can be categorized into three kinds,those methylate N6 of adenine,those methylate N4 of cytosine and those methylate C5 of cytosine.All the known DNA methyltransferases are regarding S-adenosyl methionine as the methyl donor in the methylation process.More and more evidence proved that the activity of DNA methyltransferase is intimately connected with human health.Therefore,it is beneficial to understand the various DNA methylation modes in different cells and to quantitatively analyse the DNA methyltransferase activity.Furthermore,aberrant activity of DNA methyltransferase is related to disorders and disease such as cancers.Consequently,the detection of the activity of DNA methyltransferase may eventually open the door to rountine assay and molecular diagnostics of disease.The outlook of DNA methyltransferase activity assay centainly remains positive breakthroughs and clinical applifications of DNA methyltransferase assays are realizable in the future.In view of this,in this paper we make use the entropy-driven no-enzyme amplification for real-time monitoring the process of DNA methylation and the activity of DNA methyltransferase,meanwhile,high-throughput screening of DNA methyltransferase inhibitors can also be achieved;The Guanine-rich DNA strand can form a kind of four-stranded structure known as G-quadruplex,once it binds with hemin(an anionic iron(III)porphyrin),it can exhibit peroxidase catalytic activity,and is named as G4-DNAzyme.In addition,methylated cytosine can enhance the catalytic activity of G-quadruplex.Making use of the fact that DNAzyem can catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonate disodium salt(ABTS2-)by H2O2 to produce the visual colored ABTS·-,a novel and label-free strategy was developed based on the enhancement effect of methylated cytosine.1.Based on the entropy-driven toehold-mediated hairpin displacement(ETHDA),a facile and sensitive one-step fluorescence sensing platform has been proposed for the evaluation of DNA methylation process catalyzed by methyltransferase(MTase).In this designed system,a hairpin probe is methylated by DNA adenine methylation(Dam)MTase and cleaved by DpnI endonuclease successively,liberating a catalyzer strand to initiate the signal amplification.The fluorescence intensity is increasing upon the catalyzer strand triggered circulating procedure of the ETHDA process.It is proved that the proposed biosensor is free of intricate procedures and avoids the interference of added amplification enzymes.According to the obtained result,the novel assay is exceedingly sensitive and selective in MTase detection with a low detection limit of 0.01 U·mL-1 and a wide linear range of 0.01-100 U · mL-1,which indicates that this method is a good candidate for monitoring DNA methylation.Moreover,this biosensor can offer practical applications in the high-throughput screening of MTase inhibitors.2.G4-DNAzyme has the peroxidase activity,and its activity varies when the Guanine-rich DNA sequence is different.First,we use the complementary DNA strand to block the G-rich sequence part of DNA in order not to form the structure of G4-DNAzyme.When adding the displacement strand,the G-rich sequence is released in the free condition so that G-quadruplex can spontaneously be formed.Then the G-quadruplex binds to hemin and catalytic ABTS2-in the existence of H2O2.The chromogenic reaction is more effective and efficient when specific site of G-rich sequence is methylated by CpG methyltransferase M.SssI.The detection limit is 0.1 U ·mL-1 and the wide linear range is from 0.1 to 100 U ·mL-1.This approach is facilely operated and free of label,which can also be visualized.It provided an opportunity for diagnosis of early cancer and therapy.