MODIFIED NUCLEOSIDES AS TOOLS FOR MOLECULAR BIOLOGY

一种核苷类似物，它可以与所有的 核酸双链体中的天然碱基将得到广泛的应用， 分子生物学的工具。作为聚合酶的引物组分 在链式反应（PCR）中，可以使用通用（或通用）核苷 在核酸扩增技术中， 随机DNA池作为蛋白质工程的方法。1.设计， 衍生的核苷类似物的合成和理化性质 五元环杂环化合物的反应。从分子 在建模研究中，选择靶分子， 具有最小的双链体或三链体结构的天然核酸 螺旋的扭曲。大多数类似物含有五元 环杂环，其具有能够氢键合的取代基， 每个核酸碱基具有不同的构型。主 这项研究的目的是寻找核苷类似物， 在涉及DNA的常用分子技术中的通配符 扩增、复制和测序。2.寡核苷酸将是 构建其中修饰的核苷掺入在 连续和非连续的多个站点。的影响 双链体结构上的修饰将通过Tm测量来研究 和核磁共振光谱学。3.修饰的核苷亚磷酰胺和 将制备三磷酸和CPG连接的修饰核苷， 提供给合作者以测试它们在各种系统中的效用。 4.含五元杂环的合成寡核苷酸 嘌呤/嘧啶模拟物将作为PCR反应的引物进行分析。 这些杂环中的每一个都被设计成采用潜在的 构象，允许氢键识别所有四个 天然存在的嘌呤和嘧啶碱基。5.核苷酸 模拟嘌呤和嘧啶识别的三磷酸 碱基将被用作各种DNA聚合酶的底物。

A nucleoside analogue which could base pair equally well with all of the natural bases in a nucleic acid duplex would find extensive application as a tool for molecular biology. As a primer component for the polymerase chain reaction (PCR), a universal (or wildcard) nucleoside could be used in nucleic acid amplification techniques including the generation of randomized DNA pools as a method for protein engineering. 1. The design, synthesis, and physicochemical properties of nucleoside analogues derived from five-membered ring heterocycles will be studied. From molecular modeling studies, target molecules were selected which would pair with natural nucleic acids in duplex or triplex structures with minimal distortion of the helices. Most of the analogues contain five-membered ring heterocycles which have substituents capable of hydrogen bonding in different configurations to each of the nucleic acid bases. The primary goal of this research is to find nucleoside analogues that may function as wild-cards in commonly used molecular techniques involving DNA amplification, replication, and sequencing. 2. Oligonucleotides will be constructed in which the modified nucleosides are incorporated at contiguous and non-contiguous multiple sites. The effect of the modifications on duplex structure will be investigated by Tm measurements and NMR spectroscopy. 3. Modified nucleoside phosphoramidites and triphosphates, and CPG linked modified nucleosides will be prepared and provided to collaborators to test their utility in a variety of systems. 4. Synthetic oligonucleotides containing five member heterocyclic purine/pyrimidine mimics will be analyzed as primers for PCR reactions. Each of these heterocycles have been designed to adopt potential conformations that allow for hydrogen bond recognition of all four naturally occurring purine and pyrimidine bases. 5. Nucleotide triphosphates that mimic the recognition of both purine and pyrimidine bases will be used as substrates for a variety of DNA polymerases.