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Mechanistic studies of natural ribozymes and a synthetic DNAzyme Thomas, Jason M.


This thesis reports the results of experiments designed to help elucidate the catalytic mechanisms of three RNA cleaving nucleic acid catalysts (the 9₂₅-11 DNAzyme, and the hammerhead and hairpin ribozymes). A number of enzymological and chemical probing experiments were employed in this regard, which were inspired by antecedent studies of RNA cleaving protein enzymes. First, a novel affinity labeling technique for nucleic acid catalysts was developed to probe general base catalysis. The affinity labeling substrate analogues bear a 2'-bromoacetamide modification at their cleavage sites. The second mechanistic probe employed was 5'-bridging phosphorothioate (S-link) substrate analogues, in which sulphur replaces the native oxygen leaving group. Investigation of S-link substrate cleavage in the context of active site mutations provided insight into general acid catalysis. A novel, simplified method for synthesizing S-link substrates is also presented. Several other experiments provided further mechanistic insight including pH-rate profiling, pKa perturbation, and the use of nonbridging phosphorothioate substrates. Using many of the aforementioned experiments, the catalytic mechanism 9₂₅-11 DNAzyme (a synthetically modified DNAzyme that contains unnatural protein-like functional groups) was investigated in detail. The data suggest that 9₂₅-11 uses its synthetic functional groups to mimic the active site mechanism of the protein enzyme RNaseA. Affinity labeling, pKa perturbation, and S-link cleavage experiments were also applied to the hammerhead ribozyme. The affinity labeling data suggest that the deprotonated N1 position of G12 acts as a general base catalyst. The results of pKa perturbation and S-link cleavage experiments provide strong evidence for a unique general acid mechanism in the hammerhead ribozyme. Therein, metal coordination is used to acidify a 2'-hydroxyl and thereby improve its ability to act as general acid. Finally, affinity labeling was used to characterize the role of hairpin ribozyme G8 residue, which occupies a similar structural position to the hammerhead G12 residue. Affinity labeling indeed identified G8 as a potential general base in the hairpin ribozyme. The properties of the hairpin and hammerhead affinity labeling reactions are compared and the mechanistic implications are discussed.

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