Structure and Dynamics of RNA and Protein-RNA Complexes

项目摘要 该提案的主要目标是开发用于探测结构的改进方法， 通过核磁共振（NMR）光谱学和NMR的应用， 其他生物物理学方法，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许多RNA系统，包括RNA适体 药物Macugen和新发现的称为核糖开关的RNA调节元件。目标1将发展 RNA的NMR溶液结构测定的改进方法。一个重点将是 开发了用于测量残余偶极耦合（RDC）的新型对准技术。 这些RDC提供了长期的结构信息，这对于确定更多的 许多RNA采用的延伸结构。用于将顺磁性标签引入到 RNA将被开发并用于获得改善全局和局部结构的RDC。在 此外，最近开发的用于快速获取高分辨率四维NMR的方法将 应用于RNA的共振分配。目的二是研究抑制细胞凋亡的分子机制。 Macugen的生理靶点是血管内皮生长因子（VEGF）。马库根最近在 经FDA批准用于治疗湿性年龄相关性黄斑变性， 老年人失明的原因Macugen以高亲和力和特异性结合VEGF，从而阻断VEGF的表达。 VEGF与细胞表面受体的结合。多维异谱NMR将用于 确定与全长VEGF结合的Macugen和与VEGF结合的Macugen的溶液结构， VEGF的肝素结合结构域。这些结构研究将与生物化学研究相补充。 VEGF突变体的研究，以阐明导致VEGFMacugen高亲和力的特异性相互作用 复杂.目标3将采用荧光共振能量转移（FRET）和核磁共振 技术，以研究动力学和热力学的构象变化，在第二和 RNA的三级结构单分子FRET将用于探测 S-腺苷甲硫氨酸的配体结合结构域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， RNA的动力学总的来说，这一建议将导致更好地了解分子机制 RNA用来识别它们蛋白质伴侣并在它们的功能状态之间转换。

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.