基于生物正交反应的DNA脱碱基位点荧光探针的构建及其在细胞成像和寻找肿瘤标志物中的应用研究

DNA abasic site(AP site) is closely related to cancer disease, which may be an effective and reliable tumor marker. At present, the AP site probes have many disadvantages, such as high background signal and lack of sensitivity, which can not be used in cell imaging and quantitative analysis. How to construct a AP site probe with high sensitivity of detection, observing the damage and repair of intracellular DNA through cell imaging, and How to make AP site become a more effective and reliable tumor marker, all have become an important scientific problem. Based on naphthyridine recognizing abasic sites, the molecular was synthesized by naphthyridine connected with"cyclic olefin", then reacted with "four triazine" fluorescent molecules by bioorthogonal reaction and fluorescence enhanced, which was in order to construct a new fluorescence probe with high sensitivity detection of AP site. By synthesis and evaluation of small molecules, the optimal probe was selected, and then applicated in cell imaging. AP site content was analyzed not only in cancer and paracancerous tissues, but also in leukocytes of cancer patients and normal people, making the AP site be a more effective and reliable tumor marker, which provide an important basis for the early diagnosis of malignant tumors

DNA脱碱基位点（AP位点）与癌症疾病密切相关，可能成为一种有效可靠的肿瘤标志物。目前检测脱碱基位点的荧光探针存在着背景信号高、灵敏度低等不足，无法应用于细胞成像和准确定量分析。如何构建AP位点高灵敏度检测的新型探针，并通过细胞成像观测细胞内DNA的损伤与修复，以及如何使AP位点成为更有效可靠的肿瘤标志物，成为了亟需解决的重要科学问题。本项目基于萘啶对脱碱基位点的特异性识别，将萘啶分子连接“环状烯烃”，进而与“四嗪类”的荧光小分子发生生物正交反应，实现荧光极大增强，以构建一种高灵敏度检测AP位点的新型探针。通过小分子的设计、合成和功能评价，筛选出最优探针分子；并将该探针应用于细胞成像研究、癌组织和癌旁组织中AP位点含量、癌症患者和正常人白细胞内AP位点含量的统计学分析，使AP位点成为更有效可靠的肿瘤标志物，为恶性肿瘤的早期诊断及预警提供重要研究基础。

本项目主要围绕以下四个方面进行展开：1）完成了脱碱基位点DNA的合成、脱碱基位点荧光探针的合成，成功的构建了一种基于“扭曲内部电荷转移”的原理用于检测脱碱基位点荧光探针的新方法；2）基于全球化大疫情的背景，构建了一种可用于灵敏检测新型冠状病毒及其突变体的新方法，同时将该方法应用于其它多种病毒的检测如人乳头瘤病毒的准确检测。3）发现了一种新型的硼酸型荧光探针，并完成了活细胞内过氧化氢的荧光成像。4）发现了一种新型的检测亮氨酸氨基肽酶的荧光探针，并完成了活细胞内及斑马鱼内的亮氨酸氨基肽酶的荧光成像。