CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY - Resubmission 01

描述（由申请人提供）：在过去的十年中，已经很清楚，细胞包含比以前认为的更大的功能性rna多样性。这些rna中有许多会自我折叠，形成更高级的结构，催化化学反应，并与蛋白质相互作用，介导大量其他关键功能。结构基因组学的研究主要集中在蛋白质结构上，而忽略了核酸结构。在蛋白质数据库的近6万个结构中，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。实现这一目标是具有挑战性的，因为获得高质量RNA晶体存在困难。这些困难

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.