Structure and Dynamics of RNA and Protein-RNA Complexes

描述（由申请人提供）：本提案的主要目标是开发通过核磁共振（NMR）光谱探测RNA结构和动力学的改进方法，并应用NMR和生物化学方法了解RNA适体用于以高亲和力和特异性识别其蛋白质靶标的分子决定簇。国家的最先进的NMR技术将被用来研究在体外选择的RNA适体，是一种有效的和特异性的血管生成抑制剂。目的一是发展改进的核磁共振测定RNA溶液结构的方法。其中一个重点将是开发新的校准技术，用于测量残余偶极耦合（RDC）数据。这些RDC提供了长范围的结构信息，这对于延伸分子如RNA的结构确定是至关重要的。顺磁性标签的RNA将被开发和使用，以改善整体和局部结构的RNA。此外，最近开发的快速获取高分辨率四维NMR的方法将被应用于RNA的共振分配。这些方法有可能极大地促进RNA的NMR结构测定，通过允许在当前方法的一小部分时间内获得高分辨率的高维光谱。目的2研究RNA适体药物Macugen抑制其生理靶点血管内皮生长因子（VEGF）的分子机制。Macugen最近被FDA批准用于治疗湿性年龄相关性黄斑变性，这是老年人失明的主要原因。Macugen以高亲和力和特异性结合VEGF;从而阻断VEGF与细胞表面受体的结合。将使用多维异谱NMR来确定与VEGF的肝素结合结构域（HBD）结合的Macugen的溶液结构。这些结构研究将与HBD突变体的生化研究相补充，以更好地了解导致VEGF-Macugen复合物高亲和力的特异性相互作用。目的3研究一系列与HBD高亲和力结合的RNA适体。目标是鉴定识别HBD不同表面的适体。这项研究将确定不同的机制，适体采用识别其靶蛋白，并将导致更好地了解分子决定因素，细胞RNA使用识别其蛋白partners.Public健康相关性：这项建议将研究最近FDA批准的RNA适体药物，Macugen，与其细胞靶，血管内皮生长因子相互作用。Macugen用于治疗湿性年龄相关性黄斑变性，这是50岁以上人群失明的主要原因。

DESCRIPTION (provided by applicant): 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 biochemical methods to understand the molecular determinants that RNA aptamers use to recognize their protein targets with high affinity and specificity. State-of-the-art NMR techniques will be used to study an in vitro selected RNA aptamer that is a potent and specific inhibitor of angiogenesis. Aim 1 will develop improved methods for NMR solution structure determinations of RNA. One focus will be development of novel alignment techniques for measurement of residual dipolar couplings (RDCs) data. These RDCs provide long-range structural information which is critical for structure determinations of extended molecules such as RNAs. Paramagnetic tags for RNA will be developed and used to improve the global and local structures of RNAs. Additionally, recently developed methods for rapid acquisition of high-resolution four-dimensional NMR will be applied to resonance assignments of RNA. These methods have the potential to greatly facilitate NMR structure determinations of RNA by allowing acquisition of high-resolution high-dimensional spectra in a fraction of the time of current methods. Aim 2 will study the molecular mechanism of inhibition of the RNA aptamer drug, 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, which is the leading cause of blindness in the elderly. Macugen binds with high affinity and specificity to VEGF; thereby blocking binding of VEGF to cell surface receptors. Multi-dimensional heteronuclear NMR will be used to determine the solution structure of Macugen bound to the heparin-binding domain (HBD) of VEGF. These structural studies will be complemented with biochemical studies of HBD mutants to better understand the specific interactions that lead to high affinity of the VEGF-Macugen complex. Aim 3 will study a series of RNA aptamers that all bind with high affinity to the HBD. The goal is to identify aptamers that recognize different surfaces of the HBD. This study will identify different mechanisms that aptamers employ to recognize their target protein and will lead to a better understanding of the molecular determinants that cellular RNAs use to recognize their protein partners.PUBLIC HEALTH RELEVANCE: This proposal will study the recently FDA-approved RNA aptamer drug, Macugen, interacting with its cellular target, vascular endothelial growth factor. Macugen is used to treat the wet-form of Age- Related Macular Degeneration, the leading cause of blindness in people over age 50.