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光谱将用于确定结构和研究RNA分子的折叠和阳离子结合。 将研究两个系统：（1）来自烟草环斑病毒卫星的发夹状核酶和（2）氰钴胺结合RNA适体。发夹状核酶是一种50个核苷酸的催化RNA，其对于烟草环斑病毒卫星（sTRSV）RNA的滚环复制是必需的。 发夹核酶催化导致RNA底物的切割，形成2 '3'环状磷酸和5'羟基末端，如锤头状和丁型肝炎核酶所观察到的。 然而，发夹状核酶的二级结构、折叠拓扑结构和阳离子在催化中的作用与其他已知的核酶不同。 具有底物的sTRSV发夹状核酶的二级结构由两个结构域A和B组成，每个结构域含有侧翼为Watson-Crick配对螺旋的内环。 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多维多核NMR光谱方法对13 C，15 N标记的RNA确定孤立结构域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含有UV可交联的亚结构域，该亚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，最后研究完整的核酶。这项研究的结果应该补充发夹核酶的分子生物学和生物化学的现有信息，并提供新的见解RNA催化，RNA如何折叠，以及阳离子在成核折叠中的可能作用。氰钴胺（维生素B12）适体是一种体外选择的RNA，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是令人感兴趣的，因为各种生物学上重要的反应，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，并且已经提出许多这些反应最初是由RNA酶催化的。 适体的共有序列表明不寻常的假结折叠。 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如何识别和结合特异性配体提供新的见解。知识的结构应该有助于优化发夹核酶构建目前正在研究作为治疗剂对病毒感染。 2.非技术多维，多核NMR光谱将被应用于确定结构和研究RNA分子的折叠和阳离子结合。 将研究两个系统：（1）来自烟草环斑病毒卫星的发夹状核酶和（2）氰钴胺结合RNA适体。 发夹状核酶是一种50个核苷酸的催化RNA，其对于烟草环斑病毒卫星（sTRSV）RNA的滚环复制是必需的。 氰钴胺（维生素B12）适体是一种体外选择的RNA，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是令人感兴趣的，因为由RNA酶催化的各种生物学上重要的反应，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提供新的见解RNA的三级结构基序和RNA如何识别和结合特定的配体。 发夹状核酶的结构研究应补充现有的发夹状核酶的分子生物学和生物化学的信息，并提供新的见解RNA催化，RNA如何折叠，以及阳离子在成核折叠中的可能作用。此外，结构的基本知识应该有助于优化发夹核酶构建体目前正在研究作为治疗剂对病毒感染。 项目摘要 A-2