VIBRATIONAL SPECTROSCOPY OF ISOTOPICALLY LABELED NUCLEIC ACIDS

同位素标记核酸的振动光谱

• 批准号: 6120834
• 负责人: STEFAN FRANZEN
• 金额: $ 3.95万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-15 至 2000-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120834
• 关键词: biological products; biomedical resource; cardiovascular system; nucleic acids

The SIR provided [U-"C,"N]DNA (513mg in DNA 244ml TM Buffer) This project is aimed at investigating the basis for the inhibition of blood coagulation by DNA molecules. An understanding of this inhibition may lead to the development of improved methods for the regulation of blood coagulation in clinical settings such as open heart surgery. The DNA molecule of interest is fifteen residues in length and is known to bind and inhibit thrombin. The DNA molecule was found through a directed evolution search in which the binding of 10 13 distinct DNA molecules to thromb thrombin with the strongest binding to thrombin selected. The DNA molecules which bind to thrombin are referred to as aptamers. The research project is aimed at determining the structure of this DNA molecule, the determination of how this molecule binds to thrombin and the basis for the inhibition and how to use this information to guide the development of new therapeutic agents. We have determined the structure of the DNA molecule and have shown that it adopts a new structural motif for DNA which may also be important in the structure of telomere DNA. This structure, unlike the familiar double stranded duples of DNA, can only be adopted by a very limited range of DNA molecules. In a second study we have examined the properties of modified aptamers and have been able to use our structural information to predict the ability of the modified aptamers to inhibit blood clotting. In this study we have also been able to identity the binding site of the aptamer to thrombin. 15N labeled DNA will be isolated from the cells. The DNA will be converted into nucleosides via the action of nucleases and phosphatases. The individual nucleosides will be purified by HPLC. The purified nucleosides will then be converted into phosphoramidites which in turn will be used to synthesize 15N labeled aptamers. The labeled aptarners will be complexes with thrombin and multi-dimensional NMR used to determine the structure of the bound aptamer as well as aptamer-thrombin interactions.

SIR提供了[U-“ C，” N] DNA（DNA 244ml TM缓冲液中的513mg） 项目旨在调查抑制的基础 DNA分子的血液凝血。 对此的理解 抑制可能导致发展改进的方法 调节临床环境中的血液凝血的调节，例如开放 心脏手术。 感兴趣的DNA分子是15个残基 长度，已知会结合和抑制凝血酶。 DNA分子 是通过定向进化搜索找到的 10 13不同的DNA分子与最强的凝血酶凝血酶 与选择的凝血酶结合。 与 凝血酶被称为适体。 研究项目针对 在确定该DNA分子的结构时，测定 该分子如何与凝血酶结合以及 抑制作用以及如何使用此信息来指导开发 新的治疗剂。 我们已经确定了DNA的结构 分子并表明它采用了DNA的新结构基序 这在端粒DNA的结构中也可能很重要。 这 与熟悉的双链DNA的dna不同的结构只能 通过非常有限的DNA分子采用。 一秒钟 研究我们已经检查了修改的适体的特性，并具有 能够使用我们的结构信息来预测 改良的适体抑制血液凝结。 在这项研究中，我们 还能够将适体的绑定位点识别为 凝血酶。 将从细胞中分离出15N标记的DNA。 DNA 通过核酸酶的作用将其转化为核苷 磷酸酶。 单个核苷将通过HPLC纯化。 然后将纯化的核苷转换为磷光胺 这又将用于合成15N标记的适体。 这 标记为Aptarners将与凝血酶和 多维NMR用于确定结合的结构 适体和适体凝血酶相互作用。