Structure and Dynamics of RNA and Protein-RNA Complexes

RNA 和蛋白质-RNA 复合物的结构和动力学

基本信息

批准号：
8274866
负责人：
ARTHUR PARDI
金额：
$ 28.91万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-07-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8274866
关键词：
Address Affinity Age Age related macular degeneration Angiogenesis Inhibitors Angiogenic Proteins Antibodies Area Bacteriophage Pf1 Binding Binding Proteins Biochemical Biological Assay Biology Blindness Cell Surface Receptors Chemicals Complement Complex Data Deuterium Development Diagnostic Elderly Epitopes FDA approved Family Four-dimensional Fourier Transform Functional RNA Goals Health Heparin Binding Heteronuclear NMR Hot Spot Human In Vitro Kinetics Lead Maps Measurement Methods Molecular Molecular Probes NMR Spectroscopy Nuclear Magnetic Resonance Nucleic Acids Pharmaceutical Preparations Physiological Play Process Property Proteins RNA RNA Probes RNA-Protein Interaction Residual state Resolution Role Series Site Solutions Specificity Structure Surface System Techniques Testing Therapeutic Thermodynamics Time VEGF165 Variant Vascular Endothelial Growth Factors angiogenesis aptamer base cationic antimicrobial protein CAP 37 cellular targeting design genetic regulatory protein improved inhibitor/antagonist mutant novel protein complex rapid technique research study

项目摘要

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.

描述（由申请人提供）：该提案的主要目标是通过核磁共振（NMR）光谱探测RNA的结构和动力学的改进方法，以及NMR和生化方法的应用，以了解RNA APTAMERS使用具有高亲和力和特异性的蛋白质靶标的分子决定因素。最先进的NMR技术将用于研究一种体外选择的RNA适体，是一种有效且特异性的血管生成抑制剂。 AIM 1将开发改进RNA的NMR溶液结构测定的方法。一个重点是开发用于测量残留偶性耦合（RDCS）数据的新型比对技术。这些RDC提供了远程结构信息，这对于确定扩展分子（例如RNA）至关重要。将开发和用于改善RNA的全局和局部结构的RNA的顺磁性标签。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。这些方法有可能通过在当前方法的一小部分中获取高分辨率的高维光谱，从而极大地促进RNA的NMR结构测定。 AIM 2将研究抑制RNA适体药物Macugen的分子机制，其生理靶标，血管内皮生长因子（VEGF）。 Macugen最近获得了FDA批准，用于治疗与年龄相关的黄斑变性，这是老年人失明的主要原因。 Macugen与VEGF具有高亲和力和特异性结合；从而阻止VEGF与细胞表面受体的结合。多维异核NMR将用于确定与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辅相成，以更好地了解导致VEGF-MaCugen复合物高亲和力的特定相互作用。 AIM 3将研究一系列与HBD高亲和力结合的RNA适体。目的是确定识别HBD不同表面的适体。这项研究将确定Aptamers采用的不同机制来识别其靶蛋白，并将更好地理解细胞RNA用来识别其蛋白质伴侣的分子决定因素。公共健康相关性：该建议将研究最近FDA批准的RNA Aptamer药物Macugen，与其蜂窝靶标相互作用，与其细胞靶标相互作用，血管质量增长。 Macugen用于治疗与年龄相关的黄斑变性的湿式，这是50岁以上人群失明的主要原因。