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- Comparative Analysis of Intracellular Trans-Splicing Ribozyme Activity Against Hepatitis C Virus Internal Ribosome Entry Site
- Kyung-Ju Ryu Seong-Wook Lee
- J. Microbiol. 2004;42(4):361-364.
- DOI: https://doi.org/2097 [pii]
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Abstract
- Internal ribosome entry site (IRES) of the hepatitis C virus (HCV) is known to be essential for HCV replication and most conserved among HCV variants. Hence, IRES RNA is a good therapeutic target for RNA-based inhibitors, such as ribozymes. We previously proposed a new anti-HCV modulation strategy based on trans-splicing ribozymes, which can selectively replace HCV transcripts with a new RNA that exerts anti-HCV activity. To explore this procedure, sites which are accessible to ribozymes in HCV IRES were previously determined by employing an RNA mapping method in vitro. In this study, we evaluate the intracellular accessibility of the ribozymes by comparing the trans-splicing activities in cells of several ribozymes targeting different sites of the HCV IRES RNA. We assessed the intracellular activities of the ribozymes by monitoring their target-specific induction degree of both reporter gene activity and cytotoxin expression. The ribozyme capable of targeting the most accessible site identified by the mapping studies then harbored the most active trans-splicing activity in cells. These results suggest that the target sites predicted to be accessible are truly the most accessible in the cells, and thus, could be applied to the development of various RNA-based anti-HCV therapies.
- Replacement of Thymidine Phosphorylase RNA with Group I Intron of Tetrahymena thermophila by Targeted Trans-Splicing
- Young-Hee Park , Heung-Su Jung , Byung-Su Kwon , Seong-Wook Lee
- J. Microbiol. 2003;41(4):340-344.
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Abstract
- The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.
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