Research On Detection Methods Of DNA Damage Induced By Ultraviolet Irradiation

Ultraviolet（UV）irradiation mainly causes photodamage by changing the structure of DNA,thereby interrupting the replication and transcription processes of DNA in cells,which finally induces gene mutation,cell death and cancer.As the main photoproduct of DNA damage,cyclobutane pyrimidine dimer（CPD）possesses the characteristics of high content and low repair rate,serving as the criterion for the degree of DNA damage.In this thesis,two detection methods for the assay of DNA damage by UV irradiation and the subsequent photorepair process are proposed,being of important biological significance.The main contents are as follows:（1）The DNA damage and photoenzymatic repair processes are simultaneously monitored by electrochemical impedance spectroscopy（EIS）.The single-stranded d T₂₀ is damaged under UVC（254 nm）irradiation with the formation of CPD photoproducts,and the damage process is characterized by UV absorption and circular dichroism spectroscopy（CD）.The damaged d T₂₀ is assembled on the surface of the gold electrode,and the EIS signal can be amplified by the specific recognition of CPD in the damaged d T₂₀ by the CPD antibody.The EIS signal is linearly related to the damaged DNA concentration in the range of 0.005 to 0.1μM,and a detection limit of 3.06 n M is achieved.The equilibrium dissociation constant of（3.32±0.31）×10^-12 M between the damaged d T₂₀ and CPD antibody is measured by surface plasmon resonance technique,indicating the strong binding affinity.Under the excitation of UVA（365 nm）light,DNA photolyase can photorepair the CPD products in damaged d T₂₀.The repair efficiency calculated by EIS signal is 70.0%,which is consistent with the repair efficiency（71.4%）obtained by UV absorption spectroscopy.The method is sensitive and reproducible,being capable of performing quantitative analysis of DNA damage and photorepair processes.（2）Colorimetric method for the assay of UV irradiation-induced DNA damage is developed.The detection probe（DP）contains two kinds of sequences,one is adenine-rich for the synthesis of gold nanoflowers（Au NFs）,and the other partially matches the sequence of the capture probe（CP）.Using the adenine-rich sequence in DP as the template,the Au NFs are synthesized in situ,which possess high specific surface area,strong photothermal property,and excellent peroxidase-like activity.Next,another segment in the DP is hybridized to the partial sequence in the aminated CP.Hybridization of the undamaged target DNA with the exposed toehold domain in the CP triggers the occurrence of the strand displacement reaction（SDR）.Subsequently,the aminated CP/target complex is immobilized in the carboxylated microplate wells,and smaller absorbance of the H₂O₂-OPD system is attained under light conditions.The incorporation of the damaged target DNA cannot initiate the SDR.In this case,when the aminated CP/DP-Au NFs complex is immobilized in the carboxylated microplate wells,higher absorbance of the H₂O₂-OPD system is obtained under light conditions.Based on the absorbance difference of the oxidation product of OPD,quantitative analysis of the extent of DNA damage can be achieved.