Enzymology Studies In The F-8 DNAzyme

Nucleic acids are biological macromolecules that carry genetic information in living organisms.In the 1980s,Thomas Cech and Sidney Altman discovered catalytically active RNA molecules and named RNAzyme?ribozyme?.As a non-protein enzyme,RNAzyme has played an important role in the study of enzymology and the exploration of RNA function and structure.In1994,Gerald Joyce and Ronald Breaker through vitro screening techniques obtained a single-stranded DNA molecule that catalyzes RNA fragmentation.The DNA molecule that acts as a catalyst is called a DNAzyme?deoxyribozyme?.With the deepening research of its function,the exploration of structure is also increasing.The crystal structures of 10-23,9DB1 and 8-17DNAzymes have been discovered.The types of DNAzymes are increasing,and the fields of application are becoming more widespread.In recent years,the superiority of its structure has been widely used in the fields of gene therapy,biosensors and DNA origami.In 2014,Tang Zhuo researcher lab obtained a novel DNA fragmentation active DNAzyme called F-8 by vitro selction.Based on the F-8 DNAzyme sequence and secondary structure,designing the cis-structure F-8SL DNAzyme and identified various new cofactors and inhibitors.Analyzed its reaction mechanism,the cleavage site,the phosphate group and the effect on the electrophoretic migration rate.The relationship between the point mutation and the activity of the catalytic core unit was characterized.Firstly,we examined the effects of 25 compounds such as vitamins,phenols,amines and sulfhydryls on the cleavage activity of F-8SL DNAzyme.Identification of"vitamin C/Mn²⁺"combination as the best cofactor.Besides,the flavin mononucleotide?FMN?and sodium fluorescein can combine with light to enhance the catalytic activity of DNAzyme.Other new cofactors such as catechol,resorcinol,hydroquinone,pyrogallol,phloroglucinol,o-phenylenediamine,hydroxylamine and manganese ions can also promote cleavage catalytic activity.Subsequently,analyzed the fracture mechanism and examined the role of hydroxyl radical,hydrogen peroxide and superoxide anion(O₂^·–)during reaction.Using iron porphyrin-hydrogen peroxide and guanine quadruplex-iron porphyrin-hydrogen peroxide system research the role of hydroxyl radicals,found that the hydroxyl radicals did not participate in the reaction process.Using catalase?CAT?inhibition proved the hydrogen peroxide plays an important role in the cleavage reaction process of DNAzyme,and further through the combination of sodium percarbonate and"glucose/glucose oxidase"which can produce hydrogen peroxide.Added superoxide anion and superoxide dismutase to H₂O₂/Mn²⁺,vitamin C/Mn²⁺,o-phenylenediamine/Mn²⁺,hydroquinone/Mn²⁺reaction to inhibit the activity.On this basis,we speculated and verified that nitroblue tetrazolium or protein cytochrome C can also have strong inhibition of DNAzyme activity.These experimental data show that superoxide anion may play a key role in DNA oxidative cleavage.Using denaturing polyacrylamide gel electrophoresis and time-of-flight mass spectrometry study the cleavage site of F-8SR1,F-8SR2 and F-8SR3 which are designed based on F-8SL.It was identified that the F-8SL DNAzyme fragment had a phosphate group at the 5'end and a hydroxyl group at the 3'end.The single-stranded DNA carrying a phosphate group which is related to its deoxynucleotide composition,and not all of them exhibit an accelerated electrophoresis rate.Finally,single point mutation study was performed on 12 bases of the DNAzyme catalytic center,and the relationship between the mutation site and catalytic activity was detected under four different cofactors.The data showed that the deoxyribonucleotide sites of A4,T5,and C6showed high non-conservation.The non-conservative nature provides a clear remodeling site for our further design of"photocontrol active DNAzyme".In this paper,we obtained a variety of cofactors and inhibitors of F-8SL DNAzyme,proposed a possible catalytic mechanism,analyzed the cleavage sites,and identified non-conservative nucleotides in the catalytic core.The analysis of the cleavage site and the study of the catalytic core unit point mutant laid the foundation for the subsequent artificial design of a novel structural DNAzyme.