Isothermal Nucleic Acid Amplification Detection Of DNA Modifying Enzymes

DNA-modifying enzymes are widely involved in various genetic functions such as DNA replication,repair,and recombination.The misexpression of DNA-modifying enzymes may disrupt the normal genetic functions,thereby causing various human diseases（e.g.,neurodegenerative diseases,lung cancer,and lymphoma）.Therefore,accurate and sensitive detection of DNA-modifying enzymes is of great significance for environmental monitoring,anticancer drug development and clinical diagnosis.As a basic technique in biochemistry,isothermal nucleic acid signal amplification has been developed and applied to improve the detection sensitivity of DNA modifying enzymes.In this thesis,we develope two biosensors based on isothermal nucleic acid signal amplification for ultra-sensitive detection of DNA glycosylase（UDG/h AAG）andβ-glucosyltransferase（β-GT）.We have developed a target-triggered DNAzyme motor electrochemical biosensor for simultaneous detection of multiple DNA glycosylases（UDG/h AAG）.DNA repair enzymes are responsible for the repair of DNA lesions and are important disease biomarkers.This assay involves two hairpin probes for sensing uracil DNA glycosylase（UDG）and human alkyladenine DNA glycosylase（h AAG）,a Au NPs@locked DNAzyme motor which contains the locking strands and the Mg²⁺/Pb²⁺DNAzyme strands,and a magnetic bead track which contains MB/Fc-labelled Mg²⁺/Pb²⁺substrate strands.When UDG and h AAG are present,they can cleave the corresponding hairpin DNA probes to release two single-stranded DNAs（ss DNAs）which can hybridize with the locking strands to induce the liberation of Mg²⁺/Pb²⁺DNAzyme strands from the Au NPs@locked DNAzyme motor.The subsequent hybridization of Mg²⁺/Pb²⁺DNAzyme strands with their corresponding substrate strands results in the cleavage of Mg²⁺/Pb²⁺substrate strands and the simultaneous release of MB/Fc-labelled ss DNAs from magnetic bead.The released MB/Fc labels can be electrochemically detected for accurate quantification of UDG/h AAG.The detection limit was 1.3×10^-8 U m L^-1 for UDG and 7.6×10^-8 U m L^-1for h AAG.This target-triggered DNAzyme motor exhibits high sensitivity and good specificity with the capability of simultaneously measuring multiple DNA glycosylases in He La cells and screening the potential inhibitors.We developed a fluorescent-lit biosensor for the detection ofβ-GT based on T7 transcribed nucleic acid signal amplification and a spinach RNA aptamer.β-glucosyltransferase（β-GT）can specifically catalyze the conversion of 5-hydroxymethylcytosine（5-hm C）to 5-glucosylhydroxy methylcytosine（5-ghm C）,protecting the infecting viral DNA from digestion by restriction enzymes and it is closely linked to the control of phage-specific gene expression by influencing transcription in vivo and in vitro.However,the current strategies suffer from expensive equipment,laborious treatment,radioactive hazard and unsatisfactory sensitivity.Herein,we develop a new label-free Spinach-based fluorescent biosensor for sensitive detection ofβ-GT activity by 5-hm C glucosylation-initiated rolling circle transcription（RCT）amplification of tandem Spinach RNA aptamer.The 5-hm C-modified multifunctional detection probe（MDP）can induce the performance of target-recognition,Msp I digestion,transcription amplification.Theβ-GT catalyzed 5-hm C glucosylation can protect the 5-hm C MDP probe from Msp I cleavage,subsequently initiating the RCT to generate a tandem spinach RNA aptamer,lightened up by the addition of fluorescent dye 3,5-difluoro-4-hydroxybenzylidene imidazolinone（DFHBI）for label-freeβ-GT quantitative analysis.The low background signal can be achieved by inhibition of nonspecific amplification due to the high accuracy and specificity of Msp I-catalyzed cleavage of nonglucosylated DNA substrates.Due to the higher efficiency of RCT than the canonical promoter-initiated RNA synthesis,the signal-to-noise ratio of RCT is increased by about 4.6-fold compared to that of linear template-based transcription amplification（LTTA）.This method exhibits good specificity and high sensitivity with a detection limit of as low as 2.03×10^-5 U m L^-1,and it can be further applied for potential inhibitors screening and kinetic analysis,with promising applications in epigenetic research and drug discovery.