A New Method For Fluorescence Sensing Of Uracil-DNA Glycosylase

As an important initiate enzyme for base excision repair(BER),uracil-DNA glycosylase(UDG)can repair uracil-induced DNA damages to maintain the integrity of genomic.However,the aberrant expression of UDG is closely related to the occurrence and development of various human diseases,such as huran immunodeficiency,neurodegeneration,lymphoma and cancer,and UDG has hence become an important target for medical diagnosises and treatments of some complex diseases.The development of sensitive methods for detecting UDG activity is essential for basic biological research and drug discovery,but conventional methods of UDG assay are often time-consuming and insensitive.In this dissertation,we have developed two new fluorescence methods for the detection of uracil-DNA glycosylase activity based on some simple and efficient ntcleic acid amplification techniques and fluorescence detection tnethods.The main contents are as follows:I.Detection of UDG activity based on fluorescence resonance energy transfer mechanismBased on the fluorescence resonance energy transfer(FRET)property of the cationic conjugated polymer PFP(poly[(9,9-bis(6 '-N,N,N-triethy-lammonium)hexyl)fluorenylene phenylene dibromide])and combined with the UDG-induced cleavage reaction of restriction endonuclease IV(Endo IV),we designed a nucleic acid amplification-free,simple and sensitive homogeneous detection method to quantify the activity of UDG in chapter 2.When a hairpin-structured probe,which contains four uracil deoxyribonucleotides(dU)in the stem region and five thymines(T)in the loop was incubated with Endo ?,the stem-bop structure will stay unchanged since dUs are not removed.At this time,the embedded double-strand DNA specific fluorescent dye SYBR Green ?(SG ?)and the fluorescent conjugated polymer PFP were added as fluorescence resonance energy transfer pair,and FRET phenomena from PFP to SG I occurred under strong electrostatic attraction.However,in the presence of UDG,the uracil bases are specifically removed by UDG and Endo IV cleavage the apurinic/apyrimidinic sites(AP sites)to generate single-stranded DNA.Taking the advantage of weaker affinity between SG I and single-stranded DNA,the FRET signal will be remarkably reduced.Therefore,quantitative detection of UDG is achieved based on the inverse relationship between the FRET efficiency and the activity of the UDG.?.Sensitive detection of UDG activity based on terminal deoxynucleotidyl transferase-assisted formation of fluorescent copper nanoclusters(CuNCs)In chapter 3,a nucleic acid amplification method based on terminal deoxynucleotide transferase(TdT)and deoxythymidine triphosphate(dTTP)was proposed,and the fluorescence copper nanoclusters(CuNCs)were used as the sensing elements to realize the sensitive detection of UDG.In this study,a uracil-containing stem-loop structure DNA was rationally designed as a substrate for UDG,and its 3' end was blocked by 2',3'-dideoxycytosine(ddC).UDG can specifically remove the uracils from the DNA substrate to produce AP sites,which can then be cleaved by Enod IV to ejq>ose the 3'-OH terminus.TdT will initiate a tenplate-free DNA extension reaction along the exposed 3'-OH end to produce a very long poly(T)tail,which will serve as a perfect template for the production of fluorescent CuNCs.By recording the fluorescence signal of CuNCs,the activity of UDG can be faithfully detected.Conversely,if UDG is absent,the 3'-ddC terminus of the DNA substrate cannot be recognized by TdT,so no TdT-based extension reaction and formation of CuNCs will occur.The use of ddC as a 3'-end blocker can greatly decrease the non-specific DNA extension and improve signal to noise ratio.In addition,TdT is a template-free,sequence-independent DNA polymerase that can efficiently catalyzes the tailing process to form poly(T)sequences with to thousands of thymines,and eachpoly(T)can form many fluorescent CuNCs,thus achieving ultra-high sensitivity.In addition,during the TdT-mediated extension,by introducing a poly(A)oligonucleotide containing an abasic site,a branch amplification mechanism was also preliminarily designed to further reduce the detection limit of UDG activity.