MODIFIED NUCLEOSIDES AS TOOLS FOR MOLECULAR BIOLOGY

一种核苷类似物，可以与所有的 核酸双链体中的天然碱基将被广泛应用于 分子生物学的工具。作为聚合酶的启动子成分 链式反应(PCR)，可以使用通用(或通配符)核苷 在核酸扩增技术中，包括产生 随机DNA池作为蛋白质工程的一种方法。1.设计， 核苷类似物的合成及其物化性质 我们将研究五元环上的杂环。来自分子 建模研究，选择了目标分子与 天然核酸以最少的双链或三链结构 螺旋的扭曲。大多数类似物都含有五元 具有能够氢键的取代基的环杂环 每个核酸碱基的不同构型。初级阶段 这项研究的目标是寻找可能具有以下功能的核苷类似物 涉及DNA的常用分子技术中的通配符 扩增、复制和测序。2.寡核苷酸将成为 其中修饰的核苷被结合在 连续和非连续的多个站点。美国政府的影响 将通过TM测量来研究对双链结构的修改 和核磁共振波谱。3.修饰的核苷亚磷酰胺和 将制备三磷酸盐和CPG连接的修饰核苷，并 提供给协作者，以测试其在各种系统中的实用性。 4.含五元杂环的合成寡核苷酸 将分析嘌呤/嘧啶模拟物作为聚合酶链式反应的引子。 这些杂环中的每一个都被设计成采用潜在的 允许识别所有四种氢键的构象 天然的嘌呤和嘧啶碱基。5.核苷酸 能同时识别嘌呤和嘧啶的三磷酸盐 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.