IN SEARCH OF THE UNIVERSAL NUCLEIC ACID BASE

寻找通用核酸碱基

• 批准号: 3304979
• 负责人: Donald E. BERGSTROM
• 金额: $ 19.62万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304979
• 关键词: chemical structure function; hydrogen bond; imidazole; messenger RNA; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid hybridization; nucleic acid probes; nucleobase analog; oligonucleotides; phosphamide; polymerase chain reaction; pyrimidine dimers; site directed mutagenesis; thermodynamics; triazoles

Modified nucleosides designed to function as universal nucleic acid bases will be synthesized and their suitability as constituents of oligonucleotide probes determined in physicochemical and molecular biological studies. The candidate structures include 2-carbamoyl-4-beta-D-deoxyribofuranosylimidazole, 5-carbamoyl-3-beta-D-deoxyribofuranosyl-1,2,4-triazole, and 6-amino-4-pyrimidone linked to deoxyribose via three atom spacers between C-5 and C-l'. The compounds are designed to base-pair through two hydrogen bonds to dA, dC, dG, or T. The four-fold degeneracy is obtained by constructing molecules wherein independent rotation of two bonds is sufficient to shift both base position and hydrogen bond donor and acceptor sites. The specific studies to be pursued include the following: 1) The nucleosides will be synthesized and incorporated into oligonucleotides by way of standard phosphoramidite chemistry. 2) The melting curves and thermodynamic properties of representative oligonucleotides containing the universal bases will be determined. 3) The ability of universal base modified oligomers to function in hybridization assays for the detection of low levels of mRNA will be determined. 4) Oligomers containing the universal base will also be assessed for their ability to serve as sequencing primers, as primers in the polymerase chain reaction (PCR). 5) Finally, experiments will be done to determine whether oligonucleotides which contain the modified bases can be used as templates for site-directed mutagenesis. It is clear that a universal base would have extensive and direct application to biochemistry and molecular biology. Its impact on molecular genetics, and biomedical research could be profound.

设计用作通用核酸碱基的修饰核苷 将被合成，并将它们作为 在理化和分子生物学中测定寡核苷酸探针 生物学研究。候选结构包括 2-氨基甲酰基-4-β-D-脱氧呋喃核糖基咪唑， 5-氨基甲酰基-3-β-D-脱氧呋喃核糖基-1，2，4-三唑，和 6-氨基-4-嘧啶酮通过三个原子间隔区与脱氧核糖连接， C5和C1这些化合物被设计成通过两个氢原子进行碱基配对， 与dA、dC、dG或T成键。四重简并度由下式获得： 构建其中两个键的独立旋转 足以移动碱基位置和氢键供体和受体 网站.具体研究包括：1） 核苷将被合成并掺入寡核苷酸中， 标准亚磷酰胺化学方法。2)熔化曲线和 含有该化合物的代表性寡核苷酸的热力学性质 将确定通用基础。3)万能底座的能力 修饰的寡聚体在用于检测的杂交测定中起作用 低水平的mRNA将被确定。4)低聚物含有 通用基地也将评估他们的能力， 测序引物，如聚合酶链式反应（PCR）中的引物。第五章） 最后，将进行实验以确定寡核苷酸是否 含有修饰碱基的分子可用作定点模板 诱变显然，一个普遍的基础将具有广泛和 直接应用于生物化学和分子生物学。Its impact on 分子遗传学和生物医学研究可能是深远的。