CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY - Resubmission 01

伴侣辅助 RNA 晶体学 - 重新提交 01

• 批准号: 8506004
• 负责人: Joseph Anthony Piccirilli
• 金额: $ 33.13万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506004
• 关键词: Address; Affinity; Antibodies; Antibody Formation; Antibody Repertoire; Antigens; Area; Back; Beds; Benchmarking; Binding; Biology; Catalytic RNA; Charge; Clear Cell; Complex; Crystallization; Crystallography; Databases; Development; Disease; Epitopes; Fab Immunoglobulins; Family; Functional RNA; Future; Generations; Goals; Health; Higher Order Chromatin Structure; Human; Immune; Immune system; Individual; Knowledge; Libraries; Mediating; Molecular Chaperones; Molecular Weight; Nucleic Acids; Nucleotides; Phage Display; Phase; Play; Probability; Procedures; Process; Production; Proteins; RNA; RNA Binding; RNA Folding; RNA Ligase (ATP); RNA analysis; RNA-Binding Proteins; Reagent; Recombinants; Research; Ribonucleoproteins; Role; Source; Specificity; Staging; Structure; Surface; System; Technology; Testing; Therapeutic; Tissues; Vision; Work; base; chemical reaction; combinatorial; design; design and construction; feeding; functional group; high throughput technology; inorganic phosphate; next generation; novel; novel strategies; nucleic acid structure; protein complex; protein structure; public health relevance; stem; structural genomics

DESCRIPTION (provided by applicant): In the last decade it has become clear that the cell contains a greater diversity of functional RNAs than previously thought. Many of these RNAs fold back upon themselves to form higher order structures, catalyzing chemical reactions and interacting with proteins to mediate a plethora of other critical functions. Structural genomics initiatives have focused on protein structure and all but ignored nucleic acid structure. Of the nearly 60,000 structures in the Protein Data Bank fewer than 3% are nucleic acids. Given the obvious value of structure in understanding biology and disease and in developing therapeutic strategies, RNA structure determination cannot continue at its current pace, and future structural genomics initiatives must necessarily address this bottleneck. Achieving this goal is challenging because of the difficulties associated with obtaining high quality RNA crystals. These difficulties stem in part from factors that confound lattice formation including the mutually repulsive negatively charged phosphates that decorate the surface of an RNA and the lack of diverse functional groups for mediating crystal contacts. In this application, we propose a potentially transformative approach to RNA crystallography in which we use recombinant Fab (antigen binding fragment) technology to develop an integrative pipeline for application of chaperone assisted RNA crystallography (CARC). Because traditional approaches for antibody production are not amenable to complex RNA targets, powerful immunomethods, including Fab assisted crystallography, have generally been orthogonal to RNA research. We will circumvent this problem by developing phage display libraries tailored for efficient generation of Fabs that bind to RNA, and we will use the Fabs to facilitate crystallization and structure determination of a selected test bed of high-hanging RNAs and ribonucleoprotein complexes (RNPs). Another major benefit from our efforts will be the development of a rich source of next-generation affinity reagents for functional analysis of RNA and RNPs and novel "designer" RNA binding proteins.

描述（由申请人提供）：在过去的十年中，已经清楚细胞含有比之前想象的更多的功能性RNA多样性。许多这些 RNA 自身折叠形成更高级的结构，催化化学反应并与蛋白质相互作用以介导大量其他关键功能。结构基因组学计划主要关注蛋白质结构，而几乎忽略了核酸结构。在蛋白质数据库的近 60,000 个结构中，只有不到 3% 是核酸。鉴于结构在理解生物学和疾病以及制定治疗策略方面的明显价值，RNA结构测定不能继续以目前的速度进行，未来的结构基因组学计划必须解决这一瓶颈。实现这一目标具有挑战性，因为获得高质量 RNA 晶体存在困难。这些困难 部分源于干扰晶格形成的因素，包括装饰 RNA 表面的相互排斥的带负电的磷酸盐以及缺乏介导晶体接触的多种官能团。在此应用中，我们提出了一种潜在的 RNA 晶体学变革方法，其中我们使用重组 Fab（抗原结合片段）技术来开发用于分子伴侣辅助 RNA 晶体学 (CARC) 应用的综合管道。由于传统的抗体生产方法不适用于复杂的 RNA 靶标，因此强大的免疫方法（包括 Fab 辅助晶体学）通常与 RNA 研究正交。我们将通过开发专为有效生成与 RNA 结合的 Fab 而定制的噬菌体展示文库来规避这个问题，并且我们将使用 Fab 来促进选定的高悬 RNA 和核糖核蛋白复合物 (RNP) 测试床的结晶和结构测定。我们努力的另一个主要好处是开发下一代亲和力的丰富来源 用于 RNA 和 RNP 功能分析的试剂以及新型“设计”RNA 结合蛋白。