芘二聚体发夹探针用于细胞DNA单链断裂修复能力的评估  

Direct Evaluation of Single-strand Break Repair Capacity ofCells Using a Pyrene Excimer-based Hairpin Probe

作　　者：李印 刘劲松[1] 王禹[1] 祁婷婷 孟乐园 张晶[1] 吕冬梅[1] 焦虎平[1] LI Yin;LIU Jinsong;WANG Yu;QI Tingting;MENG Leyuan;ZHANG Jing;LYU Dongmei;JIAO Huping(College of Animal Science,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物科学学院,长春130062

出　　处：《高等学校化学学报》2023年第11期8-16,共9页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金(批准号:31501909)资助。

摘　　要：基于芘分子二聚体的荧光特性设计了一种新型的发夹探针,用于评估不同细胞的单链断裂修复(SSBR)能力.首先利用芘荧光分子设计一个茎上有缺口的DNA发夹探针,该探针在Bst DNA聚合酶作用下可以水解,使荧光信号“关闭”.如果探针茎上的缺口被DNA修复酶修复,可以阻止Bst DNA聚合酶对探针的消化作用,从而阻止荧光信号的“关闭”.该设计可以用于评估细胞的SSBR能力,并通过提取细胞的核蛋白考察了不同细胞的SSBR能力.研究结果表明,肿瘤细胞及细胞系不具有原代细胞的SSBR能力.利用SSBR的关键酶进一步探索了肿瘤细胞SSBR能力缺失的原因,证明是由于肿瘤细胞中含有可以抑制SSBR过程的活性物质.此外,该方法能够同时进行500个细胞的SSBR能力检测,并用于抗衰老药物筛选.A novel pyrene excimer-based hairpin probe with a nick at the stem was developed to directly evaluate the single-strand break repair(SSBR)ability.In the existence of appropriate activity of DNA repair-associated enzymes(DREs),the oligonucleotide probe could prevent the digestion of Bst DNA polymerase and keep the long-wavelength excimer signal.However,in the absence of DREs,the nicked probe could be digested by Bst DNA polymerase,bringing about a“turn-off”monomer fluorescence signal.Therefore,the fluorescence changes of the probe could be used to directly evaluate the SSBR capability.After feasibility verification and a series of conditions optimization,the assessment of SSBR capacity by nucleoprotein extracted from different cells was investigated and the results showed that,compared to the primary cell,the tumor cell lines do not have the ability of SSBR.We also explored the defi-ciency of tumor cells SSBR by compensation with extra SSBR key enzymes and the results indicated that something might inhibit the SSBR in tumor cell lines.Furthermore,the reporter system could detect SSBR of 500 cells and was successfully applied to anti-aging drug screening.The advantages of this method include simple procedure,less time,and good repeatability,and this method can be used for the rapid detection of SSBR capacity of different cells.

关 键 词：单链断裂修复 芘二聚体 发夹探针 原代细胞 肿瘤细胞 

分 类 号：O657.39[理学—分析化学]