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Villarimo, Cynthia A.

Microbiology and Molecular Genetics

2004

Ph. D.

The focus of this dissertation is to address the intracellular problems that the hairpin ribozyme faces and propose potential solutions. A ribozyme is a catalytic RNA that is capable of targeting via sequence recognition and cleaving an RNA substrate. The catalytic activity and sequence recognition makes the ribozyme a potential antiviral therapeutic. A ribozyme was designed to target nucleotide 8242 in the Sindbis virus genomic and subgenomic RNA. The transcription of this ribozyme is driven by the human tRNAval promoter which allows for cytoplasmic localization. In addition, a Sindbis viral packaging sequence has been incorporated downstream of the ribozyme sequence, which will permit for co-localization of the ribozyme with a newly packaged virus. in vitro cleavage assay have shown that the tRNA-8242 ribozyme folds properly and is active. Primer extension assays indicate that the ribozyme are expressed in transfected cells. Results from fluorescent in situ hybridization assays show that the ribozyme is localized in the cytoplasm of transiently transfected cells. The preliminary results from plaques assays show that at 12 hours post infection, stable cell lines expressing the tRNA-8242 ribozyme have a 92% inhibition of Sindbis virus replication.
Another promoter system, the adenoviral VAl, has been employed to drive the expression of the 8242 ribozyme and was compared to the tRNAval promoter. The kinetic results show that there is a greater activity in ribozymes expressed under the tRNAval promoter. In addition, another ribozyme targeting nucleotide 277 of the Sindbis virus genomic RNA, was selected for via in vitro selection method and tested. The kinetic results showed that the 277 ribozyme had low activity in an intracellular environment.