新型疏水性非天然碱基对的设计合成及其DNA复制活性研究

Unnatural base pairs are a class of novel nucleotide derivatives, which can complete replication, transcription and translation independently from natural base pairs. Unnatural base pairs have attracted great interest in the field of nucleic acid probe, PCR technology, encoding unnatural amino acids, etc. However, the current unnatural base pairs still suffer from low efficiency and fidelity, which limit their further applications in vivo and in vitro. We have discovered that TPT3 base could significantly improve both of efficiency and fidelity during the replication of this kind of hydrophobic unnatural base pair in the previous research. Therefore, in this project, with TPT3 as leading compound, based on the mode of interaction in the replication of hydrophobic base pair, we will design, synthesize and try to find a new unnatural base structure with higher replication activity, and then develop a novel natural-like unnatural base pair. Furthermore, we can functionalize our target unnatural base pair through incorporation of linker and ribosylation, and exploit new functionalized nucleotides for PCR-compatible nucleic acid labelling technology. During this procedure, using the TPT3 derivatives as chemical biology probe, we will also investigate the effect of geometrical shape, charge distribution and other physical chemical properties on the molecular interaction procedure involved in the replication of this kind of hydrophobic base pair.

非天然碱基对是一类新近发现的能够正交于天然碱基对进行复制、转录和翻译蛋白质的核苷酸衍物。它在核酸探针、PCR技术以及编码非天然氨基酸等方面展现出诱人的前景。现有的非天然碱基对存在复制效率低和识别忠实性差等缺陷，提高非天然碱基对的复制效率和识别忠实性是推动其发展和应用的关键问题。针对该问题，本项目以前期获得的具有良好活性的TPT3碱基作为先导结构，依据疏水碱基作用方式，通过引入取代基、调整杂原子种类和位置等手段对其进行结构修饰，以期获得具有接近天然碱基对复制能力的新型疏水性非天然碱基对。进而，对所获的具有最佳复制性能的碱基对进行功能衍生化，通过引入连接臂和核糖基化策略，开发PCR兼容的可用于核酸定点标记技术的新型功能核苷酸单体。在此过程中，利用所设计合成的非天然碱基核苷酸作为化学生物学工具，揭示该类疏水碱基的几何形状、静电分布等因素在复制过程中的分子作用机制。

非天然碱基对（Unnatural Base Pair）是一类新近发现的能够正交于天然碱基对进行复制、转录和翻译蛋白质的核苷酸衍物。设计合成结构多样的非天然碱基对是研究非天然碱基作用机理、开发具有高活性、高特异性核酸探针、核酸适配体以及制备半合成生物体的重用途径。鉴于此，本项目在前期获得的TPT3碱基的结构基础上，通过设计合成结构类似物考察了TPT3母体结构修饰对RNA聚合酶识别作用的影响，发现了全新的RNA聚合酶识别非天然碱基的构效关系；通过考察连接臂的种类对聚合酶活性及后修饰方法的影响设计合成了三类连接臂TPT3碱基类似物，并成功应用于双标记16S-rRNA探针的制备和SELEX技术；通过建立特测定非天然碱基体内保留度（retention）的方法，研究了TPT3母体结构与已有的非天然碱基体内DNA复制效率，最终发现TPT3母体碱基结构无论是在DNA序列适应性还是体内复制保留度上均表现出最佳效果。