BIOORGANIC STUDIES ON LARGE STRUCTURED RNAS

大型结构 RNA 的生物有机研究

• 批准号: 2182044
• 负责人: Alanna Schepartz
• 金额: $ 20.03万
• 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 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182044
• 关键词: RNA; RNA binding protein; antisense nucleic acid; antiviral agents; chemical binding; chemical cleavage; chemical kinetics; chemical synthesis; conformation; crosslink; double stranded RNA; ethylenediaminetetraacetate; genetic regulatory element; magnesium; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; nucleic acid structure; ribonucleoproteins; synthetic nucleotide; triple helix; virus infection mechanism

This application describes general strategies for the recognition of structured RNAs, and the continued development of methodology for the mapping of higher-order RNA structure. The ability to design molecules that bind discrete RNAs with high affinity and specificity has major implications for the treatment of oncogenic and viral disease. The ability to determine experimentally the tertiary fold of an RNA molecule is a critical first step in the design of inhibitors active at the RNA level. This application focuses on the recognition, higher-order structure, and protein-binding of the Rev Response Element (RRE), a 234 nucleotide RNA located within the env coding region of the HIV-1 genome. The RRE interacts with the viral protein Rev in the nucleus of infected cells to regulate the cytoplasmic appearance of mRNAs encoding viral structural proteins. The RRE is a clear target for antiviral therapy as well as fundamental research on the interactions of RNAs with proteins, with ligands, and with themselves. A new class of molecules (Tethered Oligonucleotide Probes, TOPs) constructed during the last period bind RNAs on the basis of both sequence and tertiary structure. TOPs consist of two short oligonucleotides linked by a flexible, synthetic tether. Here is proposed an in vitro selection experiment to screen DNA and RNA "sequence space" for nucleic acid tethers that replace the synthetic tether and increase the stability of the TOP:RRE complex, the rate of its formation, or both. Such molecules should be preorganized for binding, or interact with the RRE through tertiary interactions. The proposed research may lead to the identification of DNA and RNA sequences that expand the nucleic acid structural repertoire, and could lead to new strategies for antisense inhibitors. A second strategy for accommodating RNA structure is proposed in which a TOP targets a single- and a double-stranded region; the single- stranded region via duplex formation and the double-stranded region via triplex formation. With methodology developed during the last period, molecules will be synthesized that contain the RNA cleavage agent EDTA.Fe(II) joined to a single, internal site within the RRE. Incubation of the modified RREs with a reductant will induce RNA self-cleavage at sites close in tertiary space to the attachment site. With these modified RREs, we will examine whether proposed base-pairing interactions within the stem II region are present in the free RRE or are induced by Rev binding, how these changes depend on Mg2+, how conformation within stem II changes when RRE binds Rev, and how conformation changes when RRE binds RBP9-27. An analogous experiment using structurally well-characterized tRNA(phe) will be performed to establish guidelines for data interpretation. The prediction of RNA structure from sequence is impeded by a lack of understanding of tertiary interactions. In addition to furthering our understanding of protein:RNA interactions, this research should allow identification of tertiary interactions that stabilize the RRE.

该应用程序描述了认可的一般策略 结构化的RNA，以及持续发展的方法论 高阶RNA结构的映射。设计分子的能力 具有高亲和力和特异性的离散RNA的结合主要 对治疗致癌和病毒疾病的影响。能力 为了实验确定RNA分子的第三倍倍是A 在RNA水平上活跃的抑制剂设计的关键第一步。 该应用程序着重于识别，高阶结构和 Rev反应元件（RRE）的蛋白质结合，234个核苷酸RNA 位于HIV-1基因组的ENV编码区域内。 RRE 在感染细胞的核中与病毒蛋白Rev相互作用 调节编码病毒结构的mRNA的细胞质外观 蛋白质。 RRE是抗病毒治疗的明确目标 关于RNA与蛋白质相互作用的基础研究， 配体和自己。一类新的分子（束缚 寡核苷酸探针，上一个期间构建的顶部）结合RNA 基于序列和三级结构。顶部由两个组成 短寡核苷酸由柔性，合成的系带连接。这是 提出了一个体外选择实验，以筛选DNA和RNA序列 替代合成系绳和的核酸系的空间 提高顶部的稳定性：RRE复合物，其形成速率， 或两者兼而有之。此类分子应预组织或相互作用 通过第三级相互作用进行RRE。拟议的研究可能会导致 鉴定DNA和RNA序列的扩展核。 酸性结构曲目，可能导致反义的新策略 抑制剂。提出了适应RNA结构的第二种策略 其中顶部靶向单链区域和双链区域；单人 通过双工形成和双链区域滞留区域通过 三层形成。在最后一个时期开发的方法学， 将合成包含RNA裂解剂的分子 EDTA.FE（II）加入了RRE内的一个内部站点。孵化 带有还原剂的改性RRE将诱导RNA自我切割 站点在三级空间附近的站点到附件站点。使用这些修改 RRES，我们将检查是否提出的基础配对相互作用 茎II区域存在于自由RRE中或由Rev诱导 约束，这些如何改变 取决于mg2+，当RRE结合时，茎II内的构象如何变化 转速，以及当RRE绑定RBP9-27时构象如何变化。类似的 使用结构良好的tRNA（PHE）实验将是 执行以建立数据解释准则。预测 缺乏对序列的RNA结构的影响 三级互动。除了进一步了解我们对 蛋白质：RNA相互作用，这项研究应允许鉴定 稳定RRE的三级相互作用。