A strategy for the determination of in vivo activity of a hairpin ribozyme
Hoenle, Robert J.
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Over the past ten years science has seen a gradual development of a new technology which involves the use of catalytic RNA molecules termed ribozymes. These unique forms of RNA have been shown to accurately and efficiently cleave heterologous RNAs in vitro and evidence is now emerging that may show that ribozymes can function in an in vivo enviroment. The purpose of this study is to present evidence of in vivo ribozyme activity on the phenotypic and molecular level. A ribozyme of the hairpin type was incorporated into the genome of Chinese hamster ovary cells along with its target, the chloramphenicol acetyl transferase gene, via calcium phosphate precipitation. A sensitive assay was developed using the endonuclease Si and radioactive probes to search for the presence of the ribozyme, the presence of CAT mRNA and the expected cleavage products. The outcome of the experiment was hindered by the unexpected instability of CAT mRNA driven by the promoter used in this study. The stable existence of the ribozyme however was confirmed.