Structured RNA sequences play direct roles in many viral diseases. Any effort to combat established or emerging viral threats (such as SARS, Dengue, Yellow Fever, Hanta, West Nile, HCV, etc.) must include RNA structural studies as part of a combined, multi-approach strategy. Structured viral RNAs drive or regulate processes such as viral translation initiation, programmed ribosomal frameshifting, viral RNA replication, and packaging. In many cases, a highly conserved structured viral RNA is known to be necessary for viral pathogenesis, but the precise purpose of the structure is unknown. More RNA structural data will allow us to generate testable hypotheses for the role and mechanism of these RNAs, and will provide insight into how their function could be altered. Hence, structural information is a critical input for modern antiviral development efforts. The number of unsolved viral RNA structures greatly outnumbers those that have been solved, a gap that is due in part to the technical challenges of solving RNA structures by X-ray crystallography. Our goal is to develop technology that will be the foundation for broad-based viral RNA structural genomics efforts. The two key bottlenecks that limit our ability to solve RNA structures by crystallography are: (1) the rate at which RNA can be purified for crystallization trials, and (2) the rate at which suitable heavy atom derivatives for phasing can be identified. We will develop new, high-throughput methods to address these key technical challenges. This will fundamentally transform RNA X-ray crystallography in the same way that similar methods transformed protein crystallography. First, we will develop a universal affinity-based approach for purification of large quantities of RNA that will allow structural quantities of RNA to be purified by a single affinity column method in less than 6 hours. Second, we will identify a motif that can be introduced into any RNA sequence with no structural perturbation and that will reliably bind a heavy atom for phasing based on anomalous scattering methods. Incorporation of this phasing module into an RNA sequence at the beginning stages of crystallization trials will result in a guaranteed derivative when initial crystals are obtained. Application of these two technologies will change the way we approach viral RNA crystallography and the way we approach development of new antiviral therapies.

Viruses such as SARS, West Nile, Hanta, Dengue, Hepatitis C, and many others are continuing serious threats to human health. This research aims to develop new tools to transform our ability to determine the three-dimensional structure of a multitude of RNA molecules necessary for viral infection and pathogenesis. By solving the structures of these RNAs, we will inform and enable new strategies for developing therapeutics aimed at curing viral disease.

R03AI072187

2007-06-01

2009-05-31