Studies On Regulation And Application Of Nucleic Acid-based Catalytic Reaction

For a long time,nucleic acids have generally been considered as a carrier of genetic information for living organisms.In 1994,researchers demonstrated that nucleic acids have catalytic capabilities.With intensive research,more and more catalytic nucleic acids have been discovered,which can catalyze various types of reactions,including cleavage of RNA/DNA,depurination,phosphorylation,ligation of DNA,porphyrin metallation,the Diels-Alder reaction.In physiological processes,catalytic nucleic acids not only make DNA structure diverse,but also may induce gene mutation and evolution.Catalytic nucleic acids have the advantages of easy modification,low price,high stability and strong specificity.They have been widely used in the field of biosensors.In this thesis,We conducted nucleic acid-catalyzed reactions of DNA self-catalyzed depurination and Cu²⁺-dependent DNAzyme cleavage.The main contents are as follows:1.Fluorescently probing site-specific and self-catalyzed DNA depurinationDepurination occurs via hydrolysis of the purine-deoxyribose glycosyl bond and causes nucleic acid damage.In particular,the DNA sequences that can undergo a self-catalyzed depurination?SCD?will cause a great uncertainty in duplicating,separating,purifying,and storing the DNA samples.Therefore,there is a great demand to develop a rapid detection method for SCD events.Herein,the use of a convenient fluorescence method to follow the site-specific SCD was demonstrated.We found that the resultant apurine site?AP site?from depurination can be selectively recognized by a fluorescent probe of palmatine?PAL?with a turn-on fluorescence response.The dependence of SCD on the bases of the depurination site,pH,metal ions,and time shows that our method can be used to rapidly evaluate the depurination process.Furthermore,the depurination process can be photo-switched using a photoacid as an external initiator.Our work will find wide applications in preliminarily identifying the DNA depurination.2.Fluorescently label-free Cu²⁺-dependent DNAzyme cleavageTraditional detection of metal ion-dependent DNAzyme cleavage requires modification of DNA,which is not only time-consuming but also expensive.In this thesis,we demonstrated the feasibility of using chelerythrine?CHE?to follow Cu²⁺-dependent DNAzyme.Triplex of Cu²⁺-dependent DNAzyme combines with CHE which produces strong fluorescent signal.After the addition of Cu²⁺and sodium ascorbate,the cleavage of the substrate chain leads to the disintegration of the triplex and the fluorescence decrease.Based on this unlabeled DNA fluorescence cleavage detection method,we tested effects of pH,Cu²⁺concentration,SA concentration,catalytic time and substrate chain sequence.In addition,this method has high selectivity for Cu²⁺-dependent DNAzyme,which provides new possibilities for the construction of metal ion sensing platforms.