无金属人工核酸酶的设计合成和DNA的定位切割

项目名称： 无金属人工核酸酶的设计合成和DNA的定位切割

项目编号： No.20872061

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 陆国元

作者单位： 南京大学

项目金额： 30万元

中文摘要： 人工核酸酶在分子生物学和新药研究开发有重要应用。文献报道的人工核酸酶大部份是过渡金属配合物，它们一般通过自由基机理切割核酸，因而限止了它们在分子生物学和药物中的应用。为了克服这一缺点，本项目设计合成鉴定了一系列无金属人工核酸酶- - - - 插入基-切割基偶联物。它们以三氮杂冠醚(TACN)、三氮杂冠醚原甲酰胺(TACNOA)、双胍基和环胍基为切割基，蒽醌、吡啶等平面芳环为插入基，不同长度碳链为桥链。用凝胶电泳、ESI-MS、CD光谱、荧光光谱和紫外-可见光谱等研究了这些人工核酸酶与DNA的相互作用，对DNA的切割效率、切割反应动力学和反应机理。结果表明插入基平面芳环和六个碳脂肪链为桥链的人工核酸酶能大幅度提高切割速度。其切割机理是磷酯转移机理或水解机理。特别是蒽醌-双(三氮杂冠醚原甲酰胺)偶联物，由于两个邻近的电正性的TACNOA单元的协同催化作用和蒽醌环的插入作用，在生理条件下表现最佳的切割能力，动力学数据表明符合米氏方程，具有酶催化特征，kmax为 0.17 h-1 ,是DNA未催化的10000000倍。细胞内生物活性研究表明也具有较强的抗癌活性(IC50 =1.8 ×0-6 M)。

中文关键词： 人工核酸酶;DNA切割;双核协同催化;无金属催化;电泳

英文摘要： The artificial nucleic acid nuclease has potential applications in the fields of molecular biological technology and new drug development. Metal complexes as cleaving agents of nucleic acids have been widely investigated, but their pharmic use is hampered by concerns over the lability and toxicity due to the free radical generation during the redox processes of some transition metal. Based on the structure of natural nuclease, we designed and synthesized a series of free-metal artificial nuclease compounds, in which the triazacrown ether(TACN)，tacnorthoamide (TACNOA),bisqunidine and cycloqunidine, the anthraquinone and pyridine groups, C6 alkyl were employed as cleavaging groups, intercalator of DNA, proper spacer, respectively. The calf thymus DNA binding behavior of the free-metal artificial nuclease was investigated by fluorescence, UV and circular dichroism (CD) spectroscopy. The cleavage efficiency,kinetics and mechanisms for the cleavage of supercoiled DNA by the free-metal artificial nucleases were investigated by gel electrophoresis analysis and ESI-MS. The results indicate that DNA cleavage efficiency promoted by the artificial nucleases with intercalator exhibit remarkable increases. Especiely, the conjugates of anthraquinone and two tacnorthoamide (tacnoa) units connected via C6 alkyl spacers fit to a Michaelis-Menten-type equation with kmax of 0.17 h-1 giving 10000000-fold rate acceleration over uncatalyzed DNA. The acceleration is driven by the cooperative catalysis of the two tacnoa units and the anthraquinone moiety intercalating into DNA base pairs via stacking interaction, and the transphosphorylation and hydrolysis are the possible mechanisms for the DNA cleavage. The investigation about biological activity indicate that it has strong anti-cacer action in cell(IC50 =1.8×-6 M).

英文关键词： artificial nuclease; DNA cleavage; binuclear synergistic catalysis; metal-free catalysis; electrophoresis