OLIGONUCLEOTIDES AS PROBES OF RNA AND RIBOZYME STRUCTURE

寡核苷酸作为 RNA 和核酶结构的探针

• 批准号: 2182043
• 负责人: Alanna Schepartz
• 金额: $ 18.12万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182043
• 关键词: RNA; X ray crystallography; aminoacyl tRNA; chemical cleavage; crosslink; enzyme mechanism; enzyme structure; macromolecule; messenger RNA; molecular shape; nuclear magnetic resonance spectroscopy; nucleic acid hybridization; nucleic acid probes; nucleic acid structure; oligonucleotides; phosphodiesterases; protein folding; ribonucleoproteins; ribonucleotides; ribosomal RNA; ribozymes; yeasts

The objective of this proposal is to develop a new method (linked- oligonucleotide probes) for the analysis of RNA structure in solution. To do this, the strengths of chemical synthesis and enzymology have been combined in a technique which is expected to be more effective than either crosslinking or chemical probes. This approach requires the synthesis of molecules that contain two short oligonucleotides, covalently linked, by a flexible tether of defined length. One of the oligonucleotides will be constructed of normal deoxynucleotides, the other, of 2'-OMe ribonucleotides. Each will be complementary to a single-stranded region present in the RNA under investigation. Simultaneous hybridization of both ends of this molecule to RNA will occur only if the flexible tether that separates the two oligonucleotides can traverse the complementary sites. Because of the substrate specificity of RNase H, treatment of the hybrid with this enzyme will cleave the RNA exclusively at the site of the RNA:DNA hybrid. Determination of the lengths of the RNA fragments generated will identify the cleavage site to nucleotide resolution. Through the use of tethers of varying length, the distance in space between the two complementary sites on the macromolecule can be defined. Because the linked-oligonucleotide probes can be synthesized either entirely or predominantly using automated solid phase methods, this technique will be more versatile than conventional crosslinking. Because the data can be interpreted only in terms of tertiary structure, this technique will be more straightforward than chemical or enzymatic higher-order structural analysis. This application will focus on the use of this technique to investigate the structure of native RNA molecules in solution. However, the methods described can be applied to the study of ribonucleoprotein structure, as well as RNA:protein and RNA:DNA interactions in solution. Specific Aims 1 and 2 focus on the development of this technique using structurally well characterized yeast tRNAphe. In the first of these Specific Aims, the 2'-OMe pentanucleotide 5'-UmGmGmUmGm, complementary to the 3'-end of tRNAphe, will be linked by an abasic phosphodiester tether to tetraoligonucleotides complementary to either the anticodon, D- or T-loops. By targeting these three loops, we will define a relationship between the length of the tether and the distance in A units. In Specific Aim 2, the two oligonucleotides will be linked by a poly (diaminopentane) linker. Electrostatic effects may enhance the affinity of these probes for RNA, resulting in superior molecular rulers. In Specific Aim 3, the techniques developed in Specific Aim 1 and 2 will be applied towards the study of the three dimensional structure of a catalytic ribozyme. Our goal is to use this information to determine which residues constitute the enzyme active site..

该建议的目的是开发一种新方法（链接 - 寡核苷酸探针）用于分析溶液中RNA结构。 到 这样做，化学合成和酶学的优势已经 结合一项比任何一个都更有效的技术 交联或化学探针。 这种方法需要合成 包含两个短寡核苷酸的分子，通过A 定义长度的柔性系绳。 其中一种寡核苷酸将是 由正常的脱氧核苷酸，另一个由2'-ome构建 核糖核苷酸。 每个都将与单链区域互补 存在于正在研究的RNA中。 同时杂交 该分子的末端仅在柔性系绳时才发生 分离两个寡核苷酸可以穿越互补位点。 由于RNase H的底物特异性，杂种的处理 使用该酶将仅在RNA的位点裂解RNA：DNA 杂交种。 确定生成的RNA片段长度将 确定裂解位点以分辨率。 通过使用 长度不同，两者之间的空间距离 可以定义大分子上的互补位点。 因为 可以完全合成链接的 - 寡核苷酸探针或 主要使用自动固相方法，该技术将是 比传统的交联更通用。 因为数据可以是 仅根据三级结构来解释，该技术将是 比化学或酶高阶结构更直接 分析。 此应用程序将重点介绍该技术的使用 研究溶液中天然RNA分子的结构。 然而， 所描述的方法可以应用于核糖核蛋白的研究 结构以及RNA：蛋白质和RNA：溶液中的DNA相互作用。 具体目标1和2专注于使用此技术的开发 结构良好的酵母菌特征。 在第一个 具体目的，2'-ome pentanucleotide 5'-umgmgmgmgm，互补 trnaphe的3'末端将通过无碱性磷酸二酯系列连接到 四核苷核苷酸辅助与反密码子，D-环或T环相互互补。 通过针对这三个循环，我们将定义 系绳的长度和单位的距离。 在特定的目标2中 两种寡核苷酸将通过聚（二氨基戊烷）接头连接。 静电效应可能会增强这些探针对RNA的亲和力， 导致上分子统治者。 在特定目标3中，这些技术 在特定目标1和2中开发的将用于研究 催化核酶的三维结构。 我们的目标是使用 这些信息以确定哪些残基构成活性酶 地点..