特异性识别DNA损伤的新型分子印迹纳米材料的设计、合成及应用

Genotoxic carcinogens, including chemotherapeutic anticancer agents, environmental pollutants and their metabolites，can covalently react with nucleophilic sites in DNA and result in DNA damages. It is an arduous task to specifically detect trace DNA lesions in the presence of large excess of unmodified nucleotides. The objective of this project is to fabricate a molecularly imprinted polymer nanocomposites (nanoMIP) as the artificial antibody for detection of DNA damages, including three benzo[a]pyrene(B[a]P)-DNA adducts and one oxidative product thymidine glycol (TG). A new polymerization strategy of initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP) is employed for tuning the morphology and distribution of the binding-sites of the nanoMIPs. Based on these novel artificial antibodies, liquid chromatography-mass spectrometry, immuno-capillary electrophoresis- laser-induced fluorescence and fluorescently imaged particle counting techniques are established for highly sensitive detection of DNA damages isolated from cultured cancer cells. Compared with traditional MIP particles, nanoMIP antibodies exhibit their unique properties: homogenous distribution of binding sites, high yield of the high-affinity sites (hundreds of nM, 100-fold enhanced affinity to the conventional ones), and good site accessibility which enables a fast binding kinetics. This highly-sensitive detection of DNA lesions may offer a promising alternative to immuogenic antibodies-based immunoassays for genomics and DNA modification analysis. The highlights of this study are expected to be 1) the uses of tailor-made functional monomers and ICAR-ATRP strategy for nanoMIP synthesis, 2)the new-developed methods based on nanoMIP artificial antibodies for detection of trace DNA damages in cacer cell.

DNA损伤分析对于疾病临床诊断和有毒化学品风险评估具有重要意义，其面临的突出挑战是如何在细胞大量正常DNA分子中精确甄别并检测出痕量DNA损伤。本项目根据分子印迹原理，采用引发剂连续再生催化剂-原子转移自由基聚合技术制备几种高选择性分子印迹纳米材料（nanoMIP）用作苯并[a]芘-DNA加合物、氧化应激产物TG等损伤的人工抗体；发展基于nanoMIP的色谱-质谱、免疫荧光毛细管电泳和激光共聚焦荧光成像计数分析方法，用于细胞基因组中痕量DNA损伤产物的高灵敏分析。通过特殊设计的功能单体和DNA损伤探针，使得nanoMIP的亲和力提升到几百nM水平，采用新的活性/可控自由基聚合技术，显著改善其结构和识别位点的均一性以及识别动力学特征，这些是对分子印迹方法理论和应用的拓展和创新。本项目研制的人工抗体有望作为天然抗体的补充或替代用于DNA损伤的免疫分析，亦具有一定应用价值。

项目旨在研发用于DNA损伤产物（苯并(a)芘-DNA加合物、氧化应激产物TG等）特异性识别和高灵敏分析的分子印迹纳米材料，作为“人工抗体”用以在细胞大量正常DNA碱基中精确甄别并检测出痕量DNA损伤。研究取得如下进展和发现：1）制备了苯并(a)芘代谢活性产物BPDE的DNA加合物（PBDE-dG）的分子印迹纳米材料（nanoMIP）。通过设计功能单体和采用ICAR-ATRP活性聚合技术，获得了nanoMIP对靶分子具有较高的亲和力 (880 nM) ，同时识别位点分布均一性等热力学和动力学性能得以显著提高。发展了基于nanoMIP 免疫激光共聚焦荧光成像计数分析方法，用于A549细胞基因组中痕量DNA 损伤产物的高灵敏分析，灵敏度可达18 pM。2）以设计的两种硼酸衍生物作为功能单体制备了氧化应激产物TG 的人工抗体nanoMIP，既可用于免疫激光共聚焦荧光成像计数分析方法直接检测基因组DNA中TG损伤的含量，也可以用作选择性萃取材料提取DNA样品中的TG损伤并结合液相色谱-质谱高灵敏分析。3）为解决传统DNA酶切技术速度慢、易受水解和氧化干扰的困境，制备了基于固定化酶技术的DNase I-SVP-ALPase三酶级联酶反应器，可将细胞基因组DNA在45 min内完全酶切为单核苷（酶切效率>99.7%），提高速度近20倍。结合HPLC-MS/MS技术，发展了DNA氧化损伤产物8-oxodG的快速酶切、高灵敏分析的新方法。4）利用以上新发展的样品富集和检测方法，对121份癌症患者和健康志愿者血液中的氧化损伤产物进行了测定，发现癌症患者血液中8-oxodG水平普遍高于健康志愿者。已发表SCI论文4篇，申请专利1件，培养研究生8人；基本完成项目的各项任务和指标，达到了预期目标。所发展的技术和方法为DNA损伤的分析提供了新的策略和途径，具有重要的方法学意义和较好的实际应用价值。