Base-Excision Repair-based Fluorescent Biosensing Strategies For The Detection Of Uracil-DNA Glycosylases Activity

Uracil-DNA glycosylase(UDG)is an important kind of uracil damage repair proteins.It can remove a uracil from DNA by identifying the uracil in DNA and catalyzing cleaving the N-glycosidic bond between uracil and deoxyribose,leaving an abasic site(AP).It can start the base excision repair path,and then combine with other protease to complete the whole process of DNA damage repair.It plays an important role in the base excision repair and maintains the integrity of the genome.In view of the above analysis,we have developed base excision repair-based fluorescence biosensing method for sensitive and accurate detection the activity of UDG.This paper is divided into three chapters.Chapter 1 is an introduction.In this section,It outlines the cause and the mechanism of base excision repair,the structure and function of UDG,the significance of the study and the excising detection methods of UDG activity.Further more,the problem of UDG activity assay are also summarised.In chapter 2,a sensitive and accurate method for UDG activity detection was developed on the basis of self-primer and self-template recycle rolling circle amplification(Self-RRCA)strategy.First,an immature template(IT)with a uracil base was designed,which could hybridize with a designed primer to form a pre-amplicon probe(PA probe).Under the action of UDG and endonuclease IV(endo IV),making the PA probe form a RCA amplicon through reconformation.The RCA amplicon subsequently was used to trigger the RCA,and after Nt.BbvCI nicking reaction,new amplicons were released to initiate next RCA,constituting a Self-RRCA.In this method,the designed IT was not fully complementary with the primer in the ligation part,which could effectively avoid nonspecific ligation reaction and eventually effectively avoid nonspecific amplification.Compared with the linear RCA,the Self-RRCA exhibited higher amplification efficiency.Due to above advantages,a sensitive and accurate detection method was achieved with a limit of 5×10-5 U mL-1.Furthermore,the method was adopted to screen the inhibitor of UDG and assay the activity of UDG in HeLa cell lysate.This method will offer a promising analysis tool for further biomedical research of UDG and clinical diagnosis.In chapter 3,an endogenous enzyme-initiated DNA walker for detection of UDG activity in living cells was developed.First,the gold nanoparticles were decorated with substrate strand(SR)and DNA walker strand(DW).A fluorophore FAM was labeled at the 3' end of SR and an uracil base was designed in the SR.Due to the fluorescence resonance energy transfer,the fluorescence of FAM in the SR had been quenched by gold nanoparticles.Second,a part of the DW was complementary to the SR.Under the action of UDG,the uracil base was removed,leaving an AP site.Then with the help of human apurinic/apyrimidinic endonucleases(APE1),the AP site was cut off and the fluorescence of FAM-labled SR was recovery after being cut off.With the hybridzation of DW and the next SR,an enhanced fluorescence signal was produced.In this proposed strategy,first,the operation of DW is based on the intracellular endogenous enzyme,without the addition of protease and any other substance,which ensures the normal operation of DW in the cell.Next,due to the increase of local concentration of DNA in gold nanoparticles,the enzyme cutting reaction and unloading of SR can be further promoted.This is conducive to the rapid reaction of DW and the release of fluorescence.Finally,Because the DNA strand are loaded onto the gold nanoparticles,it can effectively avoid the degradation of the DNA chain by intracellular nucleases.This strategy will provide an important tool for detecting other DNA repair enzymes in cells.