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.
RNA and RNA-protein complexes (RNPs) play a major role in human health and disease. Our knowledge of the structural basis for RNA function is severely limited by difficulties in obtaining high quality crystals of RNA and RNP complexes. We propose a new approach to RNA crystallography that unleashes the power of antibodies as crystallization chaperones for RNA and RNP complexes.
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