The broad long-term objective of this work is to characterize the local structural and dynamical features of RNA molecules, particularly those which regulate of biological function. This is consistent with the key hypothesis of Structural Biology that the three-dimensional arrangements of biomolecules are intimately related to their function. There is also the implicit understanding that structural change, expressed rigorously in thermodynamic and kinetic terms, is vital to understanding biological processes. During this project period we will use NMR to characterize specific RNA substructural units, and and the interactions of these substructures in larger, biologically relevant assemblies. The primary biological targets planned for this project are the binding site for the coat protein of R17 virus, and substructures from the 5 prime-leader sequence of HIV-1 involved in the packaging signal for the virus. Our primary aims for this project period are to: (1) Refine the ~wild- type~ variant of the structure of a 24-nucleotide R17 viral RNA using NMR distance and angle constraints. (2) Synthesize and use NMR to analyze RNA hairpins corresponding to substructures of the packaging signal in the HIV-1 genome. Hypothesis for their 3D folding will be tested by constraining their structures with very stable base-pairing blocks in the stems. (3) Use quantitative footprinting to establish the 2D folded structures of HIV-1 leader hairpins, both individually and as a part of larger superstructures. (4) Specifically label segments of the HIV-1 packaging signal with 13Carbon and 15N, and begin the process of determining its 3D structure. The whole HIV packaging signal may comprise 120nt. Even with full 13C and 15N labeling the spectrum would be difficult to interpret. Labeled hairpins will be ligated into unlabeled versions of the whole packaging signal. The key targets of our studies are subdomains of RNA viruses that known to participate in the control of processes used by viruses to package the RNA genome into infectious particles. An understanding of the structure may provide tools for designing new antiviral strategies.
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