基于G-四链体末端G平面的设计和G-四链体组装的纳米结构探究和提高 DNA过氧化物酶的催化性能

G-quadruplex and hemin could form stable complexes with peroxidase-like activity through π−π stacking. Compared with natural enzymes, such artificial enzymes offer many advantages, including low cost, high thermostability etc., which makes it important to investigate the inner mechanism of catalysis of G4/Hemin DNAzymes and improve the enzyme activity. Researches have shown that many factors, for example, the configuration of G-quadruplexes, loop species in structures and terminal residues have significant impact on the catalytic activity of G4/Hemin DNAzymes. However, the reported results only gave the initially empirical conclusions. Thus, in this study, a series of tetramolecular and intramolecular G-quadruplexes containing various kinds of terminal Guanosine-tetrads will be designed to study the binding capacity between terminal Guanosine-tetrads and hemin, and to further analyze different efficiencies of catalytic activity caused by G-quadruplex topology. Meanwhile, according to the reports in literatures, multimeric or stacked G-quadruplexes could further enhance the catalytic efficiency due to some high-activity hemin-binding sites formed between G-quadruplex units. Based on this principle, we will use some G-rich short strands to construct G4 nanostructures consisting of multiple structures, which may generate the high-activity hemin-binding sites to enhance enzyme performance. Finally, we will study the interaction between a new type of base-tetrad (GCGC-tetrad) and hemin, and further investigate the enzyme activity of GCGC-quadruplex, GCGC-tetrad contained G-quadruplexes and their nanostructures. By studying these designed discrete G-quadruplexes and G4 nanostructures, we may reveal the inner mechanism and general rules of catalysis of G4/Hemin DNAzymes, and could remarkably improve the catalytic activity of DNAzymes. This study could guide the design of the high efficient G4/Hemin DNAzymes, and promote the development of high sensitive biological and chemical detection techniques based on these high efficient G4/Hemin DNAzymes.

G-四链体与Hemin分子通过π-π堆积形成的复合物具有过氧化物酶催化作用，其具有成本低，稳定性好等显著优势，使得探究其催化的内在机理，进而提升催化活性的研究变得愈加重要。然而已有研究仅给出一些经验式结果。基于此，本项目以G-四链体末端G平面中鸟嘌呤核苷的顺反异构为出发点，设计系列末端G平面具有不同对称性和疏水性的G-四链体结构，探究这些G-四链体构型与催化性能的关系；设计多组分交替排列的G-四链体组装体纳米结构，通过堆积效应为Hemin提供更多高活性的作用位点，考察催化效率的提升；探究新型GCGC-四分体结构及其组装结构的催化性能。通过设计不同类型四链体结构，并对其与Hemin形成复合物能力进行深入研究，有望揭示G4/Hemin脱氧核酶高效催化的内在机理和一般规律，实现对其催化性能的显著提升，为建立高效的催化体系提供重要指导，同时为后续发展高灵敏生物和化学检测等打下坚实的基础。

G-四链体是由一段富G序列通过Hoogsteen氢键连接成G-四分体并堆积为具有四股核苷酸链的DNA二级结构，特定的阳离子位于结构中心进一步稳定结构。研究表明G-四链体中鸟嘌呤碱基顺反异构的堆积方式和结构中loop区域的排布对其在生物体内的功能与应用发挥重要作用。值得注意的是，基于G-四链体的结构特点，一些特异性的配体与其相互作用后显示出不同的功能性。其中G-四链体与血红素（血红蛋白的辅酶因子）形成的复合物作为一种人工酶或催化剂广泛应用在生物分析，分子机器和DNA传感器等多个领域。相较于传统的蛋白酶，DNA过氧化物酶具有成本低，易操作和稳定性好等显著优势，使得探究其催化的内在机理，进而提升催化活性的研究变得愈加重要。因此，在本项目中，我们首先通过在d(AG4A)和d(G3TG3TG3TG3)两条序列的不同位置引入3'-3'和5'-5'极性转置位点，以及对d(AG4A)中的鸟嘌呤进行溴代修饰来构建具有不同末端G-平面和不同功能基团的G-四链体，探究G-平面性质，末端碱基种类与催化活性之间的关系；之后，设计了四种“G4 +I-motif”交替连接的纳米结构，利用结构的组装调控催化功能以及结构中的界面效应来提升催化效率，这有利于构建具有可调控且高催化性能的纳米人工酶；最后，为了探究GCGC-平面对于G-四链体结构的影响以及构建新型的DNA酶或生物检测器，我们选取了之前文献中解析过的四链DNA结构，其中GCGC-四分体分布在这些结构不同的位置。之后，将它们与血红素在内不同种小分子配体分别进行孵育，探究复合物的形成效率及其产生功能的特异性。我们的研究为探究DNA过氧化物酶的内在机理提供一个新的视角，为提升酶的催化效率提供了简单有效的策略，同时对纳米检测器和检测平台的合理构建具有很好的指导作用。