RNA functions as the central conduit for information transfer in biology - in simple replicating entities like RNA viruses and in complex multi-cellular organisms. RNA is uniquely able to play this role because it encodes information at two levels: in its linear primary sequence and in its ability to form functionally critical higher-order folds. Work to date, largely focused on intensive study of a few specific regulatory motifs, has revealed that RNA secondary and tertiary structures regulate splicing, translation and protein folding, binding by small ligands and drugs and proteins, and collapse into large-scale structural domains. There are only a small number of examples in which genome-scale RNA structures have been characterized. However, numerous new biological insights were uncovered in each case. RNA viruses are especially informative systems because their compact genomes feature a dense array of functionally important secondary and tertiary structure elements. Moreover, every identification of a new regulatory motif in a pathogenic virus presents a unique target for anti-virus therapeutic design. Guided by several years of preliminary and exploratory studies, we are poised to make very high- throughput and high-content RNA secondary and tertiary structure analysis straightforward. We will apply newly invented massively parallel secondary and tertiary structure constraint-generation technologies coupled with novel molecular dynamics-driven structural refinement to understand the biological roles of higher-order structure in the hepatitis C virus (HCV) RNA genome.
Our Specific Aims are designed to reveal numerous new roles for RNA structure in the replication cycle of HCV, to do so in a way likely to inform many fields of biology, to make possible new therapeutic strategies for inhibiting viral replication, and to create tools that can be widely adopted by non-expert laboratories for analysis of complex, biologically authentic RNAs.
Aim 1 : Analyze the structure of three representative HCV RNA genomes using SHAPE, detected by massively parallel sequencing, to identify conserved base pairing and tertiary structure motifs.
Aim 2 : Create and validate an accurate and scalable approach for using experimental base pairing and through-space tertiary constraints to drive three-dimensional fold refinement for large RNAs.
Aim 3 : Integrate the technologies developed in this work to refine three-dimensional structure models and to discover new regulatory motifs for plus-sense HCV RNA genomes.
The hepatitis C virus (HCV) is a major worldwide health threat that causes fatal liver diseases and leads to two-thirds of all liver transplants and more than 50% of all liver cancers. This work will determine structures for complete HCV RNA genomes using innovative technologies created in the two collaborating project laboratories. This work is significant because it will create tools that can be widely adopted by non-expert laboratories for analysis of complex, biologically authentic RNAs;will reveal numerous new roles for RNA structure in HCV pathogenesis;and will identify novel frameworks for designing new antiviral therapeutics.
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