Catalytic activity associated with RNA molecules called ribozymes has revolutionized our understanding of biology. Although dramatic progress has been made in our knowledge of how these molecules achieve catalytic activity, many fundamental questions remain unanswered. Overall, RNA molecules must adopt a three dimensional structure assisting in the stabilization of a transition state which results in the cleavage of a unique phosphodiester bond. Presumably, to achieve the active folded structure, specific chemical groups of phosphate, sugars and nucleobase must interact to stabilize the functional three-dimensional RNA structure. Divalent cations contribute to this process by stabilizing the RNA structure and by assisting in the chemical cleavage step. The experiments outlined in this work are designed to enhance our understanding of the relationship between catalysis and structure, by focusing on the chemical groups of the RNA required to achieve catalytic activity. We have described methods which will allow us to incorporate precise modifications in the RNA molecule and to characterize their effect on the kinetics of the self-cleaving activity. In particular, we will use modified nucleotides as a surgical tool to alter chemical groups in the RNA molecule. These alterations are classified as two types. Either an exocylic group of the nucleobase is removed, usually referred to as a deletion, or a specific atom in the heterocyclic portion of the base is replaced, referred to as a substitution. To introduce these modified nucleotides into the RNA we will use either standard phosphoramidite chemistry or convert the nucleoside into a triphosphate and substitute a specific base in the RNA using a transcription reaction. To assist in the interpretation of these results, a chemical interference assay will be employed to probe the overall structure of the analogue-substituted RNA. This assay will provide a sensitive method to assess the affects of any alterations on the overall structure. Two types of chemical modifying agents will be used. The first is N-ethylnitrosourea which is directed against the phosphate backbone and the second is dimethylsulfate which is directed against nucleobase functional groups. An 89 nt fragment derived from the antigenomic RNA of the Hepatitis Delta virus will be used as the test molecule. This RNA undergoes self- cleavage, removing a 5 nt fragment from the 5' end in the presence of divalent cations. One of the attributes which makes this ribozyme unique is its ability to cleave in high concentrations of formamide. The studies described in the work will help elucidate the unusual stability associated with this RNA molecule and will help designing ribozymes as therapeutic agents for antisense technology.
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