This proposal will continue structure-function studies of the hammerhead ribozyme. The hammerhead ribozyme has been shown to efficiently cleave targeted RNAs both in vitro and in vivo. These properties have prompted many biomedical/pharmaceutical applications where ribozymes are used to try to inactivate specific gene functions by cleavage of mRNA or as antiviral agents by cleavage of the genome of RNA viruses. Additional structural data will be extremely helpful in optimizing these important biochemical applications of the hammerhead. Uniformly 13C/15N-labeled ribozymes will be synthesized by in vitro transcription. A variety of 2D and 3D multinuclear magnetic resonance experiments will be used to make resonance assignments and generate structural data on these ribozyme-substrate complexes. The NOE and J coupling data yield distance and dihedral angle information which will be used as input for molecule dynamics simulations to generate the three-dimensional structure. In addition to the structural data, dynamic information on the hammerhead will be obtained from 13C relaxation experiments. The metal binding sites in several hammerhead systems will also be probed by NMR experiments. Recently developed methods for generating partially oriented RNAs will be used to measure 1H-1H, 1H-13C, and 1H-15N dipolar couplings in the hammerhead where magnetically aligned filamentous phage lead to partial alignment of the RNA. These dipolar couplings give valuable distance and angle information that complement the standard NOE and J coupling information. A second aspect of this proposal is to understand the molecule mechanisms by which RNAs bind ligands with high affinity and specificity. We will study structure-function relations in a RNA aptamer that binds the bronchodilator theophylline with high affinity and specificity. This RNA binds theophylline with high affinity while also discriminating against the structurally similar molecule caffeine. This system shows that RNAs can be used as efficient molecule diagnostics. The structural studies will be complemented with thermodynamic and kinetic data on various mutant and modified RNAs, with the goal of understanding how this RNA aptamer recognizes theophylline with such a high level of discrimination.
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