Structure and Dynamics of RNA and Protein-RNA Complexes

项目摘要 这项提议的主要目标是开发改进的方法来探测结构和 核磁共振研究RNA的动力学及其在核磁共振和核磁共振技术中的应用 其他生物物理方法，以更好地了解RNA用于执行的分子机制 它们不同的细胞功能。我们的实验室研究了包括RNA适配子在内的许多RNA系统 药物Macugen和新发现的称为核糖开关的RNA调节元件。目标1将会发展 核磁共振溶液结构测定方法的改进。其中一个焦点将是 开发用于测量剩余偶极耦合(RDC)的新对准技术。 这些RDC提供了远程结构信息，这对于确定更多 许多RNA采用的扩展结构。用于将顺磁标签引入的通用化学 将开发和使用核糖核酸来获得改善全球和地方结构的区域发展中心。在……里面 此外，最近开发的快速获取高分辨率四维核磁共振的方法将 应用于核糖核酸的共振指认。目的2将研究其抑制的分子机制。 Macugen为其生理靶点，血管内皮生长因子(VEGF)。马库根最近 FDA批准用于治疗湿性老年性黄斑变性，领先的 导致老年人失明的原因。Macugen与血管内皮生长因子高度亲和力和特异性结合，从而阻断 血管内皮生长因子与细胞表面受体的结合。多维异核核磁共振将用于 确定与全长血管内皮生长因子结合的Macugen和与血管内皮生长因子结合的Macugen的溶液结构 血管内皮生长因子的肝素结合区。这些结构研究将与生化研究相辅相成 血管内皮生长因子突变体的研究以阐明导致血管内皮生长因子Macugen高亲和力的特定相互作用 很复杂。目标3将使用荧光共振能量转移(FRET)和核磁共振 研究次生和次生的构象变化的动力学和热力学的技术 RNA的三级结构。单分子FRET将被用来探测 S-腺苷甲硫氨酸的配体结合区和表达平台的构象状态 核糖开关。这些研究将得到核磁共振实验的补充，这些实验探索 RNA的动力学。总体而言，这一建议将有助于更好地理解分子机制 RNA用来识别它们的蛋白质伙伴并在它们的功能状态之间切换。

Project Summary The primary goals of this proposal are to develop improved methods for probing the structure and dynamics of RNAs by nuclear magnetic resonance (NMR) spectroscopy and the application of NMR and other biophysical methods to better understand the molecular mechanisms that RNAs use to carry out their varied cellular functions. Our lab studies a number of RNA systems that include the RNA aptamer drug Macugen and newly discovered RNA regulatory elements called riboswitches. Aim 1 will develop improved methods for NMR solution structure determinations of RNA. One focus will be the development of novel alignment techniques for measurement of residual dipolar couplings (RDCs). These RDCs provide long-range structural information that is critical for the determination of the more extended structures adopted by many RNAs. General chemistries for introducing paramagnetic tags into RNAs will be developed and used to obtain RDCs that improve both the global and local structure. In addition, recently developed methods for rapid acquisition of high-resolution four-dimensional NMR will be applied to resonance assignments of RNA. Aim 2 will study the molecular mechanism of inhibition of Macugen for its physiological target, vascular endothelial growth factor (VEGF). Macugen was recently approved by the FDA for treatment of the wet form of age-related macular degeneration, the leading cause of blindness in the elderly. Macugen binds with high affinity and specificity to VEGF, thus blocking the binding of VEGF to cell surface receptors. Multi-dimensional heteronuclear NMR will be used to determine the solution structures of Macugen bound to full-length VEGF and of Macugen bound to the heparin-binding domain of VEGF. These structural studies will be complemented with biochemical studies of VEGF mutants to elucidate the specific interactions that lead to the high affinity of the VEGFMacugen complex. Aim 3 will employ fluorescence resonance energy transfer (FRET) and NMR techniques to study the kinetics and thermodynamics of conformational changes in the secondary and tertiary structures of RNAs. Single molecule FRET will be used to probe transitions between conformational states for both the ligand binding domain and the expression platform of the S-adenosylmethionine riboswitch. These studies will be complemented by NMR experiments that probe the dynamics of the RNA. Overall this proposal will lead to a better understanding of molecular mechanisms that RNAs use to recognize their protein partners and to switch between their functional states.