人类端粒G-四链体碱基损伤缺失位点的选择性识别及回稳研究

This project is aimed to study the selective recognition of human telomeric DNA G-quadruplex abasic site that is produced by nucleotide damage and base loss using small bioactive molecules as the probes and the mechanism of such binding-induced G-quadruplex restabilization. The effect of position of the abasic site on the G-quadruplex stabilization and conformation will be first investigated. On the basis of this knowledge, the recognition of G-quadruplexes with differently positioned abasic site will be explored using small molecules having various substituent groups. Additionally, various experimental conditions including K+, Na+, and molecular crowding will be tested for this specific recognition. In comparison with the intact G-quadruplexes not containing the abasic site and the duplexes instead containing the abasic site, the small molecules having high binding selectivity and specificity for the G-quadruplex abasic site will be screened using electrochemical and various spectrometric technologies. Analyzing the interaction forces, the decisively structural elements of the small molecules will be identified for the recognition. The contribution of the specific recognition on the G-quadruplex stability will be clarified and the recognition mechanism will be finally established. It is well known that human telomeric DNA G-quadruplexes are closely correlated to the cell proliferation. However, the G-quadruplexes contain numerous contiguous guanine bases and thus are more easily oxidized to generate the abasic site lesion in comparison with the other DNA structures, resuliting in destabilizing the G-quadruplexes. Thus, recognizing the abasic site using small molecules is of great importance for thoroughly understanding the potential of such small molecules in tuning the G-quadruplex bioactivity and G-quadeuplex-based sensor development.

本项目研究具有生物活性的小分子对人类端粒G-四链体（G4）碱基损伤缺失产生的脱碱基位点的选择性识别及结构回稳机制。研究脱碱基位点的位置对G4稳定性及构象的影响，小分子各种环取代衍生物对G4不同位置脱碱基位点的识别及稳定化影响，以及环境条件如K+，Na+，分子拥挤等对G4结构及识别的影响。开展与结构完整的G4(不含脱碱基位点)及含脱碱基位点的ds-DNA的对比研究，采用电化学与各种谱学技术结合，筛选出对G4脱碱基位点具有高结合强度和选择性的小分子。分析识别作用力，阐明小分子对G4脱碱基位点特异性识别的关键结构要素，以及这种识别作用对G4稳定性的贡献，阐明识别原理。人类端粒G4与细胞繁殖是密切相关的。但由于G4序列中含有多个相邻的G碱基，与其它结构的DNA相比，易于氧化损伤产生脱碱基位点而使其结构失稳。因此阐明小分子的识别作用对深入理解小分子对G4活性的调制及基于G4的传感器研究有重要意义。

与其它碱基相比，核酸中鸟嘌呤G碱基最易被氧化。因此，当氧化性的空穴(hole)注入到核酸中时，G碱基最易被氧化损伤缺失而形成脱碱基位点(AP位点)。更为重要的是，从电化学角度看，由于G-四链体中含有多个相邻的G碱基，其碱基间可发生强的电子耦合作用，其氧化电位更低，因此G-四链体等富含G碱基的序列最易形成AP位点。与ds-DNA相比，G-四链体中的AP位点不易被AP内切酶识别而难于修复。本项目围绕人类端粒G-四链体、三链体等结构中AP位点导致的结构变化及选择性识别等科学问题，首先研究了非G碱基缺失而致的人类端粒G-四链体单一性hybrid-1和hybrid-2结构的探针识别特异性及机理，认为与G-四链体之外的亚结构形成有关。同时探究了G碱基缺失引起的G-四链体构型转变及失稳原因，探讨了小分子通过互补氢键及静电排斥作用选择性识别这种结构的原理。解析并获得了我们发展的特异性探针EPI识别人类端粒G-四链体的精确结构模型。研究了极性翻转位点对G-四链体结构及稳定性的影响，发现极性翻转对由两层tetrad形成的G-四链体的稳定性影响最大，甚至不能形成稳定的G-四链体结构；而由三层tetrad形成的G-四链体则构型会发生大的变化。考察了小分子识别G-四链体时对极性翻转位点依赖性，以及小分子诱导G-四链体构象变化在传感分析中的应用。此外，尝试了G-四链体与光敏剂的复合体用于构建具有光氧化能力的DNAzyme。同时开展了双链DNA碱基损伤缺失位点的识别研究，在手性分子区分、SNP分析、超分子调控、及DNA自催化等方面有潜在应用前景。最后，发展了平行双链DNA的荧光识别方法，探讨了三链体中碱基损伤缺失位点在发展新型传感器中的应用潜力。