MODIFIED NUCLEOSIDES AS TOOLS FOR MOLECULAR BIOLOGY

修饰核苷作为分子生物学的工具

• 批准号: 2392246
• 负责人: Donald E. BERGSTROM
• 金额: $ 20.25万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392246
• 关键词: DNA directed DNA polymerase; chemical models; chemical structure function; computer simulation; enzyme substrate; hydrogen bond; molecular cloning; nuclear magnetic resonance spectroscopy; nucleic acid sequence; nucleic acid structure; nucleoside analog; oligonucleotides; synthetic nucleotide

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.

一种核苷类似物，可以与所有的碱基对一样好