Study On New Cofactors Of The Pistol-like DNAzyme

DNAzyme(deoxyribozyme or catalytic DNA)is a type of single-stranded DNA molecule with catalytic activity obtained through in vitro screening.It was first reported by Breaker and Joyce in 1994.The catalytic activity of this type of enzyme mostly depends on metal ion cofactors,such as magnesium ions and zinc ions.In 1998,the Breaker research group reported a DNAzyme with the activity of oxidatively breaking DNA.It is called pistol-like DNAzyme because its secondary structure is similar to a pistol.The catalytic activity of this enzyme is dependent on the dual cofactor"X/Cu²⁺"model,the cofactor is vitamin C(VC)/copper ion(Cu²⁺)or hydrogen peroxide(H₂O₂)/copper ion(Cu²⁺).After analyzing the results of our previous research and consulting related literature,we speculated that there should be a variety of X cofactors for pistol-like DNAzymes,and 36compounds were selected(divided into 5 categories:vitamin compounds,sulfhydryl compounds,phenolic compounds,amines compounds and other compounds)are optional cofactors.When the reaction solution contained copper ions,vitamin C,reduced nicotinamide adenine dinucleotide,reduced nicotinamide adenine dinucleotide phosphate,carboxyfluorescein,reduced glutathione,cysteine,dithiothreitol,mercaptoethanol,catechol,hydroquinone,pyrogallol,phloroglucinol,dopamine,caffeic acid,o-phenylenediamine and hydroxylamine could promote pistol-like DNAzyme,it exhibited high catalytic activity and the experimental results showed that these compounds could be used as the X cofactor in the dual cofactor"X/Cu²⁺"combination.In the absence of copper ions,catechol,hydroquinone,pyrogallol,o-phenylenediamine and vitamin C could also significantly promote the enzyme's cleavage of DNA substrates,indicating that the catalytic function of the DNAzyme still existsed single cofactor dependent model.When implementing the subject of"Galvanic Reaction Regulation and Control of Pistol DNAzyme Activity",we found that the solid metal copper wire could catalyze the reaction of pistol-like DNAzyme in aqueous solution.Subsequently,the liquid phase reaction system was simplified,containing only double distilled water and pistol-like DNAzyme.Under this reaction condition,the effects of other metal copper products on the activity of pistol-like DNAzyme were further tested,and the results showed that all copper products could work,and the effect of the copper interface after polishing was stronger than the original interface.In the selective experiments of metallic copper and non-metallic samples(such as glass,ceramics and plastics,etc.),the pistol-like DNAzyme only reacted to metallic copper,showing a high degree of specificity.However,in the selective experiment of metallic materials it showed universality.When we chose 24 solid metal-action pistol DNAzyme,vanadium,copper,and tantalum had strong catalytic effects on the enzyme,followed by indium,titanium,tin,zinc,iron,niobium,aluminum,platinum and chromium,while cobalt,silver,manganese,germanium,cadmium,tungsten,gold,lead,magnesium and bismuth had weak effects.The above experimental results showed that some solid metals could be used as cofactors of pistol-like DNAzyme to participate in the catalytic reaction of enzyme.The universality of pistol-like DNAzyme for solid metal selection implied that the catalytic mechanism behind it mighe be the same.We speculated that some metals could generate reactive oxygen species(superoxide anion,hydrogen peroxide,and hydroxyl radicals)by the action of oxygen in water,and superoxide anion might be the main cause of DNA substrate fragmentation.For this reason,we conducted experiments to verify.The effects of six metal surface recovery solutions including titanium,vanadium,copper,niobium,indium,antimony,and tantalum on the activity of pistol-like DNAzyme were identified.The results showed that these solid metals might be metal ions released in water.Using copper as a cofactor,the concentration gradient experiment of ethylene diamine tetraacetic acid showed that the water contained metal ions,and the inductively coupled plasma mass spectrometer test results verified this conclusion.For the reactive oxygen species generated in the reaction system,we selected nitro-blue tetrazolium,cytochrome C,catalase and 3,3',5,5'-tetramethylbenzidine(TMB)for detection.The results showed that in addition to TMB,the other three substances could effectively inhibit the catalytic reaction,indicating that hydrogen peroxide and superoxide anion played an important role in the process of DNA fragmentation by pistol-like DNAzyme.The hydrogen peroxide concentration gradient experiment confirmed that the compound was not a key substance that mediates DNA fragmentation,but existsed as an intermediate product.This conclusion was further verified in the metal recovery liquid experiment.At the same time,oxygen elimination experiments in water showed that oxygen was an indispensable molecule for pistol-like DNAzyme reactions,and the reaction of oxygen with metals(ions)to produce superoxide anions was a key link in the enzyme's catalytic reaction.Through the above research,we developed a pistol-like DNAzyme dual cofactor"X/Cu²⁺"dependent model,identified a variety of new X cofactors and the results of the study showed that the enzyme also had a single cofactor dependent form of non-copper ions.More importantly,it had been discovered that solid metal could be used as a cofactor for the DNAzyme.In the interface reaction between metal and enzyme,the formation of superoxide anion might be a key step for pistol-like DNAzyme oxidative cleavage,which was other oxidative cleavage activity the research on the catalytic mechanism of deoxyribozyme provides reference and reference.