Characterizing RNA-metal binding by Raman spectroscopy

通过拉曼光谱表征 RNA-金属结合

• 批准号: 8016713
• 负责人: PAUL R CAREY
• 金额: $ 30.72万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8016713
• 关键词: 7SL RNA; Active Sites; Adenine; Bacteriophages; Binding; Binding Sites; Biological Process; Catalysis; Catalytic RNA; Cations; Cell Nucleus; Cells; Chemicals; Chemistry; Clinic; Clinical; Cobalt; Collaborations; Complex; Crystallography; Cytokine Inducible SH2-Containing Protein; Cytoplasm; DNA; Data; Dependency; Disease; Drug Design; Electrostatics; Endoplasmic Reticulum; Enzymes; Foundations; Frequencies; Functional RNA; Genetic Code; Goals; Guanine; Guanosine; Health; Heart; Hepatitis Delta Virus; Imidazole; Individual; Introns; Ions; Isotopes; Knowledge; Label; Laboratories; Lasers; Lead; Libraries; Life; Link; Location; Magnesium; Measures; Membrane; Messenger RNA; Metals; Methenamine; Methods; Microscope; Microscopy; Modality; Modeling; Molecular Conformation; Monitor; Mutate; Mutation; Nature; Nucleotides; Phase; Phenylalanine-Specific tRNA; Play; Population; Positioning Attribute; Process; Property; Protein Biosynthesis; RNA; RNA Binding; RNA Probes; RNA Splicing; Raman Spectrum Analysis; Reaction; Regulation; Relative (related person); Research; Resolution; Ribosomal RNA; Ribosomes; Roentgen Rays; Role; Running; Samarium; Signal Recognition Particle; Signal Transduction; Site; Small Nuclear RNA; Small RNA; Solutions; Spectrum Analysis; Stretching; Structure; Surface; System; Techniques; Testing; Time; Titrations; Translating; Vertebral column; Work; X-Ray Crystallography; analog; base; catalyst; chemical reaction; chemical synthesis; enzyme mechanism; functional group; gene function; group I ribozyme; inorganic phosphate; insight; light scattering; mRNA Precursor; novel; novel strategies; phenylalanine-tRNA; phosphodiester; phosphorothioate; programs; protonation; research study; scaffold; shape analysis; three dimensional structure; tool

DESCRIPTION (provided by applicant): RNA participates in a wide range of biological processes. In addition to its role in information transfer in the genetic code, it can act as a structural scaffold, a catalytic enzyme, or a regulatory signal. In the clinical context it is both a target for drug design and a tool for regulating gene function. Much of RNA function is driven by its interaction with metal ions and metals play a pivotal role in maintaining RNA's structure and promoting the catalytic mechanism. The principle goals of this proposal are to develop new modalities to characterize metal RNA interactions and the chemical reactions they promote. The methods involve the use of Raman spectroscopy to probe RNA in solution and Raman microscopy to study reactions in single crystals of RNA. The solution studies will use small RNA fragments, less than 40 nucleotides, in order to characterize a metal cation binding at a single phosphate group along with conformational changes in the adjacent phosphodiester backbone. The single crystal studies center on four RNAs, the Hepatitis Delta Virus (HDV) ribozyme, the P4-P6 domain and Twort ribozyme from group I introns, and phenylalanine tRNA. Magnesium binding sites in the crystal structures that have been characterized by X-ray crystallography are being used to provide the Raman signatures of -PO2- groups making inner sphere contacts with magnesium. The low frequency Raman spectral region provides the spectra, and thus chemical information, associated with the bound metals themselves. A major theme is to use Raman crystallography, combined with X-ray crystallography to provide unambiguous answers to outstanding issues of mechanism. For example, for HDV crystals we label active site G1and U-1 groups with 13C and 15N isotopes to allow us to follow these residues as catalytic parameters such as pH and Mg concentration are changed. These observations will be extended by using the labeling approach to define the chemistry of the key active site groups as the chemical reaction is being carried out in the crystal. An overarching aspect of this proposal is its interdisciplinary nature that involves collaborators at three locations. The Raman spectroscopy and Raman crystallography are undertaken in the Carey laboratory at CWRU, while the expertise and material for solution studies is provided by Drs. Mike Harris and Eric Christian from CWRU and Dr. Kwaku Dayie at the Cleveland Clinic. The work on RNA crystals is based on collaborations with Dr. Phil Bevilacqua at Penn State and Dr. Barbara Golden at Purdue. PUBLIC HEALTH RELEVANCE: Although RNA is a close relative of DNA it is far more versatile and plays many roles in the life of the cell. Thus, an understanding of the properties of RNA lies at the heart of our knowledge of the processes of life and to be able to control them when malfunction leads to disease states. The present proposal sets out to determine how RNA acts as a catalyst to bring about chemical reactions. The research involves following chemical reactions in very small RNA crystals using a laser light scattering technique.

描述（由申请人提供）： RNA参与广泛的生物过程。除了在遗传密码中的信息传递中的作用外，它还可以作为结构支架，催化酶或调节信号。在临床上，它既是药物设计的靶标，也是调节基因功能的工具。RNA的许多功能是由其与金属离子的相互作用驱动的，金属在维持RNA的结构和促进催化机制方面起着关键作用。该提案的主要目标是开发新的模式来表征金属RNA相互作用及其促进的化学反应。这些方法涉及使用拉曼光谱法来探测溶液中的RNA，以及使用拉曼显微镜来研究RNA单晶中的反应。溶液研究将使用小于40个核苷酸的小RNA片段，以表征与单个磷酸基团结合的金属阳离子沿着相邻磷酸二酯骨架的构象变化。单晶研究集中在四种RNA，丁型肝炎病毒（HDV）核酶，P4-P6结构域和来自I组内含子的Twort核酶，以及苯丙氨酸tRNA。镁结合位点的晶体结构，其特征在于X-射线晶体学被用来提供拉曼签名的-PO 2-基团，使内球接触镁。低频拉曼光谱区域提供了与结合金属本身相关的光谱，从而提供了化学信息。一个主要的主题是使用拉曼晶体学，结合X射线晶体学提供明确的答案，突出的问题的机制。例如，对于HDV晶体，我们用13 C和15 N同位素标记活性位点G1和U-1基团，以使我们能够随着催化参数（如pH和Mg浓度）的变化而跟踪这些残基。这些观察结果将通过使用标记方法来扩展，以定义晶体中进行化学反应时关键活性位点基团的化学性质。这项建议的一个重要方面是它的跨学科性质，涉及三个地点的合作者。拉曼光谱和拉曼晶体学在CWRU的Carey实验室进行，而溶液研究的专业知识和材料由CWRU的Mike Harris和Eric Christian博士以及克利夫兰诊所的Kwaku Dayie博士提供。RNA晶体的工作是基于与宾夕法尼亚州立大学的Phil贝维拉夸博士和普渡大学的Barbara Golden博士的合作。公共卫生相关性：虽然RNA是DNA的近亲，但它的用途要多得多，在细胞的生命中扮演着许多角色。因此，了解RNA的特性是我们了解生命过程的核心，并且能够在故障导致疾病状态时控制它们。目前的提议旨在确定RNA如何作为催化剂引发化学反应。这项研究涉及使用激光散射技术在非常小的RNA晶体中进行化学反应。