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

• 批准号: 8000295
• 负责人: ARTHUR PARDI
• 金额: $ 8.81万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-14 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000295
• 关键词: Address; Affinity; Age; Age related macular degeneration; Angiogenesis Inhibitors; Angiogenic Proteins; Antibodies; Area; Arts; 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; 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

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）：本提案的主要目标是开发通过核磁共振（NMR）波谱探测 RNA 结构和动力学的改进方法，并应用 NMR 和生化方法来了解 RNA 适体的分子决定因素用于以高亲和力和特异性识别其蛋白质靶标。最先进的核磁共振技术将用于研究体外选择的RNA适体，它是一种有效且特异性的血管生成抑制剂。目标 1 将开发用于 RNA NMR 溶液结构测定的改进方法。重点之一是开发用于测量残余偶极耦合（RDC）数据的新型对准技术。这些 RDC 提供长程结构信息，这对于 RNA 等延伸分子的结构测定至关重要。将开发并使用 RNA 顺磁标签来改善 RNA 的整体和局部结构。此外，最近开发的快速获取高分辨率四维 NMR 的方法将应用于 RNA 的共振分配。这些方法可以在当前方法的一小部分时间内获取高分辨率高维光谱，从而有可能极大地促进 RNA 的 NMR 结构测定。目标2将研究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与 VEGF 结合；从而阻断 VEGF 与细胞表面受体的结合。多维异核核磁共振将用于确定 Macugen 与 VEGF 肝素结合域 (HBD) 结合的溶液结构。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定相互作用。目标 3 将研究一系列与 HBD 具有高亲和力结合的 RNA 适体。目标是鉴定能够识别 HBD 不同表面的适体。这项研究将确定适体用于识别其靶蛋白的不同机制，并将有助于更好地理解细胞 RNA 用于识别其蛋白质伴侣的分子决定因素。 公共健康相关性：该提案将研究最近 FDA 批准的 RNA 适体药物 Macugen 与其细胞靶标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，这是导致 50 岁以上人群失明的主要原因。