核酸损伤的修复主要在细胞核内进行，但迄今仍缺乏能直接在活细胞的细胞核内原位观察核酸修复酶的定位和活性变化的方法，制约了对不同类型细胞核酸损伤动态修复和分子机理共同特征和相互差异的研究进程。本项目拟开发一类新颖的以含脱碱基空位DNA与RNA互补链组成的杂合链结构为基础、进一步共价偶联含核定位信号(NLS)和穿膜肽(CPP)复合肽序列的RNA/AP-DNA*-CPP<NLS>杂合荧光探针分子，将其用于不同类型活细胞的细胞核内脱碱基核酸内切酶(APE1)的原位荧光成像。进一步地，将利用荧光探针实时跟踪活细胞在不同条件下细胞核和细胞质内APE1活性的变化，指导选择恰当的时间点将细胞裂解，分别提取细胞核和细胞质中的APE1，通过质谱分析鉴定在不同空间和时间APE1的翻译后修饰，阐明调控其活性及在质-核间迁移的分子结构变化特征和关键因子。方法将用于对正常细胞和癌细胞核酸损伤修复过程的系统对照研究。

DNA damage repair mainly occurs in the nucleus of living cells. However, no tools are available now for in-situ fluorescence imaging of DNA repair enzymes in the nucleus of living cells, which limited the investigation of the dynamics of DNA repair and the differentiation between normal cells and cancer cells with regard to their DNA repair processes. In this project, we will synthesize a new type of chimeric fluorescent probe RNA/AP-DNA*-CPP<NLS> (AP=abasic site; *=labeled with fluorophore and quencher on the DNA strand; CPP=cell-permeable peptides; and NLS=nuclear localization signal) which may enter the living cells and localize in the nucleus for sensitive fluorescent response to the variation of the activity of apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1, the most important DNA repair enzyme). Using the obtained probe and the APE1 magnetic nanoprobe which has been developed in our previous project, we will track the transportation of APE1 between cytoplasm and nucleus. Guided by the real-time monitoring system, we will obtain cytoplasma and nucleus samples at important time point. Then we will isolate APE1 from these samples and characterize the post-translational modifications of APE1 by mass spectrometry. We will mainly focus on the acetylation, phosphorylation and ubiquitination of APE1 and their effects on the localization, transportation, binding to DNA substrate and activity of the enzyme, in order to reveal the major factors that regulate the functions of the enzyme. The method will be applied to compare the differences between normal cells and cancer cells in DNA damage repair processes.