Development Of Ultrasensitive Methods For The Detection Of DNA Repair Enzymes

The maintenance of genomic integrity and stability is essential for all living organisms.However,the genomes are constantly injured by various endogenous and environmental agents,creating¹0⁵ DNA lesions?e.g.,modified bases,abasic sites,DNA adducts and DNA strand breaks?in a single cell per day,leading to gene mutations and even cell death.To decrease the mutation frequency,cells have evolved multiple DNA protection mechanisms,such as base excision repair?BER?,nucleotide excision repair?NER?,homologous recombination?HR?repair,mismatches repair?MMR?,mutagenesis repair and alternative repair mechanisms,with a variety of DNA repair enzymes being involved to respond to different types of DNA damages through specific recognition and effective repair.Based on the distinct functions,DNA repair enzymes can be classified intofollowing groups:DNA glycosylases,polynucleotide kinases,phosphatases,DNA methyltransferases,DNA endonucleases/exonucleases,DNA polymerases,and DNA ligases,etc.Recent researches demonstrate that the dysregulation of DNA repair enzymes is closely related to various human diseases?e.g.,neurodegeneration,immunodeficiency,xeroderma pigmentosum,and premature aging,etc.?and cancer?e.g.,lung,liver,breast,and cervical cancers,etc.?,and DNA repair enzymes can function as an important biomarker for diseases diagnosis and potential therapeutic targets for various cancers.Therefore,the development of efficient biosensing technologies for ultrasensitive detection of DNA repair enzymes is of great significance for biochemical research and clinical diagnosis.Conventional methods for DNA repair enzymes assay includes radiolabeled-based gel electrophoresis,enzyme-linked immunosorbent assay?ELISA?,mass spectrometry?MS?and high-performance liquid chromatography?HPLC?.However,these methods have unavoidable drawbacks,such as radiation pollution,poor sensitivity,complicated manipulation and expensive instruments,limiting their practical applications.Recently,with the rapid development of scientific techniques,some new strategies including colorimetric,luminescent,electrochemical,and fluorescent assays have been developed with wide applications in the fields of chemistry,biology and medicine.Especially,the fluorescent method has distinct advantages of simple operation,high sensitivity,good selectivity and convenient signal output,and has received more and more attention.In this thesis,we have developed a series of new fluorescent methods for sensitive detection of various DNA repair enzymes including thymine DNA glycosylase?TDG?,human alkyladenine DNA glycosylase?hAAG?,polynucleotide kinase?PNK?and alkaline phosphatase?ALP?based on isothermal nucleic acid amplification and single-molecule imaging.This thesis contains the following contents:1.We have designed a new fluorescent method for real-time monitoring of TDG activity based on cyclic enzymatic repairing-mediated dual-signal amplification.Firstly,the linear probe is hybridized with the circular template,forming a circular substrate for TDG.In the presence of endonuclease IV?Endo IV?,TDG specifically cleaves the linear probe at thymine?T?site,resulting in the breaking of its 3'end sequence.The cleaved linear probe acts as a primer to initiate the rolling circle amplification?RCA?,produing long repeat sequences with uracil?U?nucleotides incorporated.The U lesions can be removed by uracil DNA glycosylase?UDG?/Endo IV-initiated base-excision to generate 3'-hydroxyl groups?3'-OHs?for new polymerization,resulting in numerous copies of reporter sequences produced.The resultant reporter sequences can subsequently hybridize with free circular templates to initiate new cycles of polymerization and U excision,eventually leading to the generation of a large number of reporter sequences.The reporter sequence hybridizes with the signal probes,triggering Endo IV-based cyclic cleavage of signal probes,generating an amplified fluorescence signal.This method has a detection limit of 5.6×10^-7 U/?L and can be applied to determine kinetic parameters and quantify TDG activity in 1 cancer cell,offering a powerful tool for genomic study,and clinical diagnostics.2.We have developed a novel fluorescent strategy for detecting hAAG activity based on controllable autocatalytic cleavage-induced fluorescence recovery.In the presence of hAAG and human apurinic/apyrimidinic endonuclease?APE1?,the 2'-deoxyinosine in hairpin probe 1?HP1?can be recognized and cleaved,leading to the unfolding of hairpin structure for forming a DNA duplex.The trigger 1 built in DNA duplex will hybridize with HP2 through the toehold-mediated strand displacement reaction?TMSDR?to induce T7 exonuclease?T7 exo?-catalyzed two-step recycling cleavage of HP2 and signal probe,resulting in fluorescence recovery.This method exhibits a detection limit of 4.9×10^-6 U/?L,and can be utilized to evaluate kinetic parameters,screen anticancer drugs and even quantify hAAG activity from 1 HeLa cell,holding great potential for biochemical and biomedical applications.3.We have demonstrated a fluorescent method for single-molecule detection of polynucleotide kinase?PNK?through phosphorylation-directed recovery of fluorescence quenched by Au nanoparticles?AuNPs?.In the presence of PNK,the?-phosphate group from adenosine triphosphate?ATP?is transferred to 5?-OH terminus,resulting in 5?-phosphorylation of the hairpin substrate.The?exonuclease??exo?will cleave the hairpin substrate from the 5'-phosphate group?PO₄?,resulting in the unfolding of hairpin structure and the formation of binding probe.The resultant binding probes may specifically hybridize with the capture probes modified on AuNPs,forming double-strand DNA?dsDNA?duplexes with 5?-PO₄.Subsequently,?exo will cleave the capture probes in dsDNA,causing the liberation of Cy5 molecules and the binding probes.The released binding probes may further hybridize with new capture probes,inducing cycles of digestion-release-hybridization and consequently the release of numerous Cy5 molecules.Through simply monitoring Cy5 molecules in the solution,PNK activity can be quantitatively measured.This assay is very sensitive with a limit of detection of 9.77×10^-8 U/?L,and it can be further used to evaluate the inhibition of adenosine diphosphate?ADP?and ammonium sulfate,and measure PNK in cancer cell extracts.More importantly,it may be used as a universal sensing platform for detecting other polynucleotide kinases.4.We have designed a fluorescent method for ALP activity detection at the single cell level based on primer dephosphorylation-initiated isothermal circular exponential amplification.Firstly,two dual-function hairpin probes?HP1 and HP2?are designed,functioning as both the templates for exponential amplification reaction?EXPAR?and the generators for signal output.In the presence of ALP,the 3?-phosphorylated primer is dephosphorylated and subsequently hybridizes with the 3?protruding end of HP1 to initiate the first strand displacement amplification?SDA?,producing trigger 1 and fluorescence signal.The released trigger 1 is complementary to the 3?protruding end of HP2 for the initiation of the second SDA,producing trigger 2 and fluorescence signal.Notably,trigger 2 is complementary to the 3?protruding end of HP1 and may initiate two consecutive SDAs,enabling circular EXPAR to convert the low-abundant ALP activity information to the exponentially amplified fluorescence signal.This method exhibits high sensitivity with a detection limit of 2.0×10^-10 U/?L,and it can be applied for the measurement of kinetic parameters,the screening of potential inhibitors,and even measure ALP at the single-cell level,providing a powerful tool for ALP-related biomedical research and clinical diagnosis.