CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY - Resubmission 01

伴侣辅助 RNA 晶体学 - 重新提交 01

基本信息

批准号：
8643797
负责人：
Joseph Anthony Piccirilli
金额：
$ 32.37万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
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/8643797
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

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

展开

项目摘要

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，很明显，该单元包含的功能性RNA的多样性比以前想象的要多。这些RNA中的许多人都向后折叠以形成高阶结构，催化化学反应并与蛋白质相互作用以介导许多其他关键功能。结构基因组学倡议集中在蛋白质结构上，几乎忽略了核酸结构。在蛋白质数据库中近60,000个结构中，核酸是核酸。鉴于结构在理解生物学和疾病以及制定治疗策略方面具有明显的价值，RNA结构的确定不能以当前的速度持续下去，并且未来的结构基因组学倡议必须必须解决这一瓶颈。实现这一目标是具有挑战性的，因为与获得高质量RNA晶体有关的困难。这些困难茎部分来自混淆晶格形成的因素，包括装饰RNA表面的互抑制性负电荷的磷酸盐以及用于介导晶体接触的不同官能团的缺乏。在此应用中，我们提出了一种潜在的转化方法来进行RNA晶体学，其中我们使用重组FAB（抗原结合片段）技术来开发用于应用伴侣辅助RNA晶体学（CARC）的集成管道。由于传统的抗体生产方法不适合复杂的RNA靶标，因此包括Fab辅助晶体学在内的强大免疫方法通常与RNA研究是正交的。我们将通过开发用于有效生成与RNA结合的Fab量身定制的噬菌体展示文库来解决这个问题，我们将使用Fab来促进结晶和结构确定高悬挂RNA和核糖核蛋白蛋白复合物（RNP）所选的测试床。我们努力的另一个主要好处将是发展下一代亲和力的丰富来源 RNA和RNP的功能分析的试剂以及新型的“ Designer” RNA结合蛋白。