Characterizing RNA-metal binding by Raman spectroscopy

描述(由申请人提供)： RNA参与了广泛的生物过程。除了它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，它既是药物设计的目标，也是调节基因功能的工具。RNA的功能在很大程度上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。这项提议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。这些方法包括使用拉曼光谱探测溶液中的RNA，以及使用拉曼显微镜研究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以及邻近磷酸二酯主链的构象变化。单晶研究集中在四个RNA，丁型肝炎病毒(HDV)核酶，第一组内含子的P4-P6结构域和Twort核酶，以及苯丙氨酸tRNA。用X射线结晶学表征的晶体结构中的镁结合位置被用来提供与镁接触的-PO2-基团的拉曼信号。低频拉曼光谱区提供与结合金属本身相关的光谱，从而提供化学信息。一个主要的主题是使用拉曼结晶学，结合X射线结晶学，为悬而未决的机理问题提供明确的答案。例如，对于HDV晶体，我们用13C和15N同位素标记活性中心G1和U-1基团，以便当pH和镁浓度等催化参数改变时跟踪这些残基。这些观察结果将通过使用标记方法来扩展，以定义在晶体中进行化学反应时关键活性位基团的化学。这项建议的一个重要方面是其跨学科性质，涉及三个地点的合作者。拉曼光谱和拉曼结晶学在CWRU的Carey实验室进行，溶液研究的专业知识和材料由CWRU的Mike Harris博士和Eric Christian博士以及克利夫兰诊所的Kwaku Daie博士提供。这项关于RNA晶体的工作是基于与宾夕法尼亚州立大学的菲尔·贝维拉奎亚博士和普渡大学的芭芭拉·戈尔登博士的合作。与公共健康相关：尽管RNA是DNA的近亲，但它的用途要广泛得多，在细胞的生命中扮演着许多角色。因此，了解RNA的性质是我们了解生命过程的核心，并能够在故障导致疾病状态时对其进行控制。本提案旨在确定RNA是如何作为催化剂引起化学反应的。这项研究涉及使用激光散射技术跟踪非常小的RNA晶体中的化学反应。

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.