Enzymology Studies In The RadDz3 Dnazyme

DNAzyme is a single-stranded DNA molecule with catalytic function,which was first reported by Ronald Breaker and Gerald Joyce in 1994.At present,it has been found that the types of catalysis of DNAzymes are mainly cleavage and ligation.Our research group mainly studies fragmented DNAzymes.Pistol-like DNAzyme?PL?is a DNAzyme with oxidative cleavage DNA activity screened by Ronald Breaker's laboratory.The enzyme activity depends on the double cofactor“X/Cu²⁺”,such as vitamin C/Cu²⁺or hydrogen peroxide/Cu²⁺.Recently we identified a number of new X cofactors for this enzyme,such as vitamin B6,coenzyme II,glutathione,pyrogallol,and hydroxylamine.These X cofactors are of different types and structures.The analysis of their catalytic mechanism may be the superoxide anion-mediated oxidative cleavage of DNA.In 2014,Tang Zhuo's group reported F-8 DNAzyme with DNA oxidative cleavage activity.Our research found that F-8 and PL can“share”most of the X cofactors,and we speculate that the catalytic mechanisms of these two DNAzymes are similar.In 2017,Scott Silverman and others accidentally obtained RadDz3 DNAzyme with oxidatively cleaved DNA activity during the screening of amide bond-cleaving active DNAzymes.This thesis takes RadDz3 DNAzyme as the research object.It is envisaged that F-8 and PL“share”X cofactors are also applicable to RadDz3DNAzyme and obtained experimental support.It is confirmed that solid metals can also participate in catalytic reactions and Cu²⁺can be used as an enzyme reaction.The co-enhancing factor transforms the double-cofactor“X/Mn²⁺”form that is dependent on the enzyme activity into the three co-factor“vitamin C/Cu²⁺/Mg²⁺(Ba²⁺/Ca²⁺)”form.At the same time,multiple types of RadDz3 inhibitors have been found.In addition,the enzyme cleavage site was identified,a DNA ligation method based on the enzyme cleavage product was proposed,and attempts were made to modify the catalytic core structure.First,we studied the RadDz3 DNAzyme's cofactor.The zymogen reaction system includes three kinds of divalent metal ions Mn²⁺,Zn²⁺and Mg²⁺.Based on the form of the double cofactor“X/Mn²⁺”of the F-8 DNAzyme,we speculate that Mn²⁺plays a major role in the catalytic reaction of RadDz3 DNAzyme.The effects of Mn²⁺,Zn²⁺,and Mg²⁺were detected using vitamin C and hydrogen peroxide.The data showed that the combination of Mn²⁺and vitamin C or sodium percarbonate had the best enzyme activity.The combination of Zn²⁺,Mg²⁺and hydrogen peroxide had certain effects,and Zn²⁺,Mg²⁺and vitamin C.The combination showed no effect,so the reaction cofactor composition could be simplified to the“X/Mn²⁺”form.Through the combined effect of hydrogen peroxide,sodium percarbonate,glucose/glucose oxidase?the latter two generate hydrogen peroxide in solution?and Mn²⁺,it is clear that hydrogen peroxide plays an important role in the catalytic process of cleavage.The combination of vitamin C/Mn²⁺was selected to optimize the conditions of vitamin C,Mn²⁺and NaCl concentration,the pH value of the reaction system,the reaction temperature and time.Under the optimized reaction conditions,the effects of 11 kinds of divalent metal ions were detected,and only Mn²⁺showed effects.In the presence of Mn²⁺,a total of 26 compounds including vitamins,phenols,amines,thiols,and fluorescence were detected using PL DNAzyme's X cofactor as a reference.NADH,fluorescein,catechol,resorcinol,hydroquinone,resorcinol,and hydroxylamine can be used as X cofactors of RadDz3 DNAzyme.Catechol derivatives such as dopamine,chlorogenic acid,and resorcinol resveratrol can play a similar role.According to the latest research in our laboratory,some solid metals can be used as cofactors for PL DNAzyme.We have tested 24 metals and found that manganese,cobalt,niobium,indium and tin can promote catalysis in the presence of hydroquinone.Of the reactions,indium metal works best,while trivalent indium ions show no effect.We have also found that Cu²⁺can effectively increase vitamin C/Mn²⁺as an enhancement factor,suggesting that Mn²⁺can act as both a cofactor and DNA folding.Based on this,we replaced Mn²⁺with Cu²⁺,which plays the role of cofactor,and replaced Mn²⁺with Mg²⁺,Ba²⁺,or Ca²⁺to assist DNA folding,and successfully constructed the vitamin C/Cu²⁺/Mg²⁺(Ba²⁺/Ca²⁺)3 cofactor form.This was followed by RadDz3 inhibitory factor study.Catalase,nitrogen blue tetrazole,and cytochrome C were selected for detection.The results showed that they could effectively inhibit the catalytic reaction,indicating that hydrogen peroxide and superoxide anions were involved in the reaction.In the process of testing whether Zn²⁺can enhance the catalytic activity of vitamin C/Mn²⁺,it was found that Zn²⁺,Co²⁺,Ni²⁺,Cd²⁺,Co²⁺,Hg²⁺,and Pb²⁺can inhibit the enzyme activity.The above metal ions could be used as inhibitors of RadDz3,and Mg²⁺,Ba²⁺and Ca²⁺did not show inhibitory effects.Glutathione,cysteine,dithiothreitol,and mercaptoethanol were highly effective X cofactors for PL DNAzyme,but experimental data showed that these compounds have a significant inhibitory effect on RadDz3 activity.The bivalent metal ion chelator EDTA and 1,10-phenanthroline had been shown to be effective in inhibiting catalytic reactions,but the reason for the inhibition of 1,10-phenanthroline may be competition for superoxide anions or binding substrates.In addition,in the research on the identification of the fracture products,it was confirmed by mass spectrometry that the newly generated 3'terminus of RadDz3 was broken with a carboxylic acid group.Based on this result,we propose a carboxyl-amino linkage reaction that can be used in an in vitro screening protocol for allosteric deoxyribozymes.Finally,we compared the secondary structure of RadDz3 and PL and tried to replace the secondary structure of the two enzymes.The experimental results did not detect the cleavage activity,indicating that the catalytic core structures of the two were conservative and not suitable for replacement and reconstruction.Through the above studies,we expanded and enriched the types and numbers of RadDz3coenzymes and inhibitors,explored their possible reaction mechanisms,analyzed the role of Mn²⁺in the reaction system,and determined the carboxyl group of the fragment.A chemical ligation method for DNA was proposed,which laid the foundation in vitro screening applications subsequently.