NMR Studies of RNA Enzymes and Aptamers

RNA 酶和适体的 NMR 研究

• 批准号: 9808072
• 负责人: Juli Feigon
• 金额: $ 33万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808072&HistoricalAwards=false
• 关键词: NMR; Studies; RNA; Enzymes; Aptamers

Feigon MCB 9808072 1. Technical Multidimensional, multinuclear NMR spectroscopy will be used to determine the structures and investigate the folding and cation binding of RNA molecules. Two systems will be studied: (1) the hairpin ribozyme from the tobacco ringspot virus satellite and (2) a cyanocobalamin-binding RNA aptamer. The hairpin ribozyme is a 50 nucleotide catalytic RNA that is essential for rolling circle replication of the tobacco ringspot virus satellite (sTRSV) RNA. Hairpin ribozyme catalysis results in the cleavage of an RNA substrate with formation of 2'3' cyclic phosphate and 5' hydroxyl termini, as observed for the hammerhead and hepatitis delta ribozymes. However, the hairpin ribozyme secondary structure, folding topology, and role of cations in catalysis are distinct from other known ribozymes. The secondary structure of the sTRSV hairpin ribozyme with substrate consists of two domains, A and B, each of which contains an internal loop flanked by Watson-Crick paired helices. The structure of domain A has been previously determined by NMR studies. The first part of this study will determine the structure of the isolated domain B, using multidimensional, multinuclear NMR spectroscopic methods on 13C,15N-labeled RNA. For the larger RNAs, specific and random, fractional deuteration is employed. Domain B contains a UV-crosslinkable subdomain which is also found in several other RNAs, and its structure will also be determined. Once the domain B structure has been characterized, the ribozyme without substrate will be studied, and finally the intact ribozyme. Results from this study should complement the existing information on the molecular biology and biochemistry of the hairpin ribozyme, and provide new insights into RNA catalysis, how RNA folds, and the possible role of cations in nucleating folding. The cyanocobalamin (Vitamin B12) aptamer is an in vitro selected RNA that binds to cyanocobalamin with high specificity and affinity (Kd ~ 90 nM). The mode of binding of the aptamer to cyanocobalamin is of interest because a variety of biologically important reactions, including the conversion of ribonucleotides into deoxyribonucleotides, are carried out by cobalamin-dependent enzymes, and it has been proposed that many of these reactions were originally catalyzed by RNA enzymes. The consensus sequence of the aptamer indicates an unusual pseudoknot fold. The structure of a minimal aptamer of 31 nucleotides which contains consensus sequence and retains high specificity and affinity to the substrate cyanocobalamin will be studied. Since this aptamer has been shown to have a specific Li+ requirement for binding, the cation dependence will also be investigated. The results should provide new insights into RNA tertiary structure motifs and how RNA can recognize and bind specific ligands. Knowledge of the structure should help in optimizing the hairpin ribozyme constructs currently being investigated as therapeutic agents against viral infections. 2. Nontechnical Multidimensional, multinuclear NMR spectroscopy will be applied to determine the structures and investigate the folding and cation binding of RNA molecules. Two systems will be studied: (1) the hairpin ribozyme from the tobacco ringspot virus satellite and (2) a cyanocobalamin-binding RNA aptamer. The hairpin ribozyme is a 50 nucleotide catalytic RNA that is essential for rolling circle replication of the tobacco ringspot virus satellite (sTRSV) RNA. The cyanocobalamin (Vitamin B12) aptamer is an in vitro selected RNA that binds to cyanocobalamin with high specificity and affinity (Kd = 90 nM). The mode of binding of the aptamer to cyanocobalamin is of interest because a variety of biologically important reactions catalyzed by RNA enzymes, including the conversion of ribonucleotides into deoxyribonucleotides, are carried out by cobalamin-dependent enzymes. The structural studies of the cyanocobalmin binder should provide new insights into RNA tertiary structure motifs and how RNA can recognize and bind specific ligands . The structural studies of the hairpin ribozyme should complement the existing information on the molecular biology and biochemistry of the hairpin riboyzme, and provide new insights into RNA catalysis, how RNA folds, and the possible role of cations in nucleating folding. In addition, basic knowledge of the structure should help in optimizing the hairpin ribozyme constructs currently being investigated as therapeutic agents against viral infections. PROJECT SUMMARY A-2

Feigon MCB 9808072 1. 技术 多维、多核核磁共振波谱将用于确定 RNA 分子的结构并研究折叠和阳离子结合。 将研究两个系统：（1）来自烟草环斑病毒卫星的发夹核酶和（2）氰钴胺结合RNA适体。发夹核酶是一种 50 个核苷酸的催化 RNA，对于烟草环斑病毒卫星 (sTRSV) RNA 的滚环复制至关重要。 发夹核酶催化导致 RNA 底物裂解，形成 2'3' 环磷酸和 5' 羟基末端，正如在锤头核酶和丁型肝炎核酶中观察到的那样。 然而，发夹核酶的二级结构、折叠拓扑和阳离子在催化中的作用与其他已知的核酶不同。 带有底物的 sTRSV 发夹核酶的二级结构由两个结构域 A 和 B 组成，每个结构域均包含一个侧翼为 Watson-Crick 配对螺旋的内部环。 结构域 A 的结构先前已通过 NMR 研究确定。 本研究的第一部分将使用 13C,15N 标记 RNA 的多维、多核 NMR 光谱方法确定分离结构域 B 的结构。对于较大的 RNA，采用特异性和随机的部分氘化。结构域 B 包含一个可紫外交联的子结构域，该结构域也存在于其他几种 RNA 中，其结构也将被确定。 一旦结构域 B 结构被表征，将研究没有底物的核酶，最后是完整的核酶。这项研究的结果应该补充有关发夹核酶的分子生物学和生物化学的现有信息，并为 RNA 催化、RNA 如何折叠以及阳离子在成核折叠中的可能作用提供新的见解。氰钴胺（维生素 B12）适体是一种体外选择的 RNA，它以高特异性和亲和力 (Kd ~ 90 nM) 与氰钴胺结合。 适体与氰钴胺的结合模式引起人们的兴趣，因为多种重要的生物学反应，包括核糖核苷酸转化为脱氧核糖核苷酸，都是由钴胺素依赖性酶进行的，并且有人提出，许多这些反应最初是由钴胺素催化的通过RNA酶。 适配体的共有序列表明存在不寻常的假结折叠。 将研究包含共有序列并保留对底物氰钴胺的高特异性和亲和力的31个核苷酸的最小适体的结构。 由于该适体已被证明具有特定的 Li+ 结合要求，因此还将研究阳离子依赖性。 这些结果将为 RNA 三级结构基序以及 RNA 如何识别和结合特定配体提供新的见解。了解该结构应该有助于优化目前正在研究的发夹核酶结构，作为抗病毒感染的治疗剂。 2. 非技术性的多维、多核核磁共振波谱将用于确定RNA分子的结构并研究其折叠和阳离子结合。 将研究两个系统：(1) 来自烟草环斑病毒卫星的发夹核酶和 (2) 氰钴胺结合 RNA 适体。 发夹核酶是一种 50 个核苷酸的催化 RNA，对于烟草环斑病毒卫星 (sTRSV) RNA 的滚环复制至关重要。 氰钴胺（维生素 B12）适体是一种体外选择的 RNA，它以高特异性和亲和力 (Kd = 90 nM) 与氰钴胺结合。 适体与氰钴胺素的结合模式令人感兴趣，因为RNA酶催化的多种重要的生物学反应，包括核糖核苷酸转化为脱氧核糖核苷酸，都是由钴胺素依赖性酶进行的。 氰钴胺结合剂的结构研究应该为 RNA 三级结构基序以及 RNA 如何识别和结合特定配体提供新的见解。 发夹核酶的结构研究应该补充有关发夹核酶的分子生物学和生物化学的现有信息，并为RNA催化、RNA如何折叠以及阳离子在成核折叠中的可能作用提供新的见解。此外，结构的基本知识应有助于优化目前正在研究作为病毒感染治疗剂的发夹核酶结构。 项目摘要 A-2