连接酶反应介导的荧光策略用于高效检测尿嘧啶-DNA糖基化酶的活性  

A Ligase Reaction-mediated Fluorescent Strategy for Highly Efficient Assaying of Uracil-DNA Glycosylase Activity

作　　者：吴玉姝 吴敏[2] 刘敏 WUYushu;WUMin;LIUMin(Institute of BioPharrnceutical Research,Liaocheng University,Liaocheng 252059,China;Liaocheng People＇s Hospital,Liaocheng 252000,China)

机构地区：[1]聊城大学生物制药研究院,山东聊城252059 [2]聊城市人民医院,山东聊城252000

出　　处：《聊城大学学报（自然科学版）》2018年第2期8-14,共7页Journal of Liaocheng University:Natural Science Edition

基　　金：国家自然科学基金项目(21473085);山东省自然科学基金项目(ZR2018LB023);山东省抗体制药协同创新中心开放课题(CIC-AD1830);聊城大学博士科研启动基金项目(318051632)资助

摘　　要：目的:灵敏检测尿嘧啶-DNA糖基化酶(UDG)活性有利于生物医学研究和疾病预后.这里,构建一种连接酶反应介导的荧光策略用于高效检测UDG活性.方法:在本策略中,两条短寡核苷酸链分别与发夹探针环部序列的一半杂交,形成含缺口的DNA复合物.在UDG作用下,发夹探针环部的单个U碱基被移除,产生无嘌呤/无嘧啶(AP)位点.AP位点能抑制缺口处的连接酶反应,使位于发夹探针末端的立足点和链迁移区域仍彼此邻近,从而引发杂交链式反应(HCR),产生大量G四倍体(G4)结构.最后,G4与N甲基卟啉IX(NMM)结合,产生增强的荧光信号.结果:本策略检测限低至0.000 20U/mL,并能区分UDG与其它DNA糖基化酶.结论:本策略为生物医学研究和疾病预后提供了一种有潜力的工具用于灵敏定量检测UDG活性.Objective：Sensitive detection of uracil-DNA glycosylase（UDG）activity is beneficial for biomedical study and disease prognosis.Here,a ligase reaction-mediated fluorescent strategy was constructed for highly efficient detection of UDG activity.Methods：In the strategy,two short oligonucleotides were separately hybridized to one-half of the loop of a hairpin probe to form a DNA complex with a nick.Under the action of UDG,only one uracil base in the hairpin-loop was removed to generate apurinic/apyrimidinic（AP）site.Accordingly,the AP site locating at the 3＇-side of the nick could inhibit the ligase reaction,leaving the toehold and branch-migration domains in the ends of hairpin probe are still adjacent.Subsequently,the adjacent toehold and branch-migration domains could initiate the hybridization chain reaction（HCR）,generating numerous G-quadruplex（G4）structures.Finally,the G4 structures could interact with N-methyl-mesoporphyrin IX（NMM）to generate enhanced fluorescent signal.Results：The detection limit was as low as 0.000 20 U/mL for UDG.In addition,UDG could be well distinguished from other DNA glycosylases.Conclusion：This strategy provided a promising tool for sensitive quantification of UDG activity in biomedical study and disease prognosis.

关 键 词：连接酶反应 尿嘧啶-DNA糖基化酶 单个尿嘧啶碱基 无嘌呤/无嘧啶位点 碱基错配 

分 类 号：O611.6[理学—无机化学]