CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY - Resubmission 01

描述（由申请人提供）：在过去的十年中，已经清楚的是，细胞含有比以前认为的更大多样性的功能RNA。这些RNA中的许多折叠回自身以形成更高级的结构，催化化学反应并与蛋白质相互作用以介导过多的其他关键功能。结构基因组学的研究主要集中在蛋白质结构上，而几乎忽略了核酸结构。在蛋白质数据库中的近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开发治疗策略方面的明显价值，RNA结构测定不能以目前的速度继续下去，未来的结构基因组学计划必须解决这一瓶颈。由于获得高质量RNA晶体存在困难，实现这一目标具有挑战性。这些困难 部分原因是混淆晶格形成的因素，包括装饰RNA表面的相互排斥的带负电荷的磷酸盐，以及缺乏介导晶体接触的多种官能团。在本申请中，我们提出了一种潜在的变革性方法，RNA晶体学中，我们使用重组Fab（抗原结合片段）技术，开发一个综合的管道应用分子伴侣辅助RNA晶体学（CARC）。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辅助结晶学，通常与RNA研究正交。我们将通过开发为有效产生结合RNA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来促进高悬RNA和核糖核蛋白复合物（RNP）的选定测试床的结晶和结构测定。我们努力的另一个主要好处将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“设计师”RNA结合蛋白。

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.