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Comparative Analysis of Intracellular Trans-Splicing Ribozyme Activity Against Hepatitis C Virus Internal Ribosome Entry Site 원문보기

The journal of microbiology, v.42 no.4, 2004년, pp.361 - 364  

Ryu Kyung-Ju (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University) ,  Lee Seong-Wook (Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University)

Abstract ▼ AI-Helper 아이콘AI-Helper

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 b...

주제어

#gene therapy   #group I intron   #HCV   #IRES   #Tetrahymena thermophila   #trans-splicing ribozyme  

AI 본문요약
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제안 방법

  • The IGS of each Zrane-splicing ribozyme is as follows: Rib86, 5- *; GAAGGA-3 Ribl95, 5*-GAGGAC; Ribl99, 5'- GAGAAA-3'; Rib251, 5'-GGCAGU-3'; Rib329, 5'- GCGAGA-3T. Intracellular Zrane-splicing activity of the ribozymes was observed by monitoring the induction rate of expression of the reporter gene, firefly luciferase (FLuc) in this study, which was tagged at the 3' end of each ribozyme. We cotransfected each ribozyme (5 |g) consisting of the specific IGS along with or without HCV IRES RNA (5 Ig) into liver-derived Huh7 cells.
  • One key factor for the application of the Zra5-splicing ribozyme to efficient HCV treatment in patients will be the identification of the most accessible sites in the target RNA. To identify and confirm the most easily targeted HCV sequence in cells in this study, we evaluated intracellular trans-splicing activities of several group I-based ribozymes which target the different sites of HCV IRES predicted to be accessible or inaccessible from RNA mapping studies in test tubes.

이론/모형

  • To determine which HCV IRES RNA uridines are accessible to ribozymes in our recent study, an RNA mapping strategy was employed in vitro (Ryu and Lee, 2003). The mapping method was based on the sequence analyses of Zrane-splicing junction sites using a ribozyme library with randomized IGS of the Tetrahymena group I intron (Lan et al., 1998; Ryu et al., 2003) and RNA tagging (Jones et al., 1996). Then, the residues between nt 191 and 199, which are present at loop Illb domain of HCV IRES (Honda et al.
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참고문헌 (15)

  1. Byun, J., N. Lan, M. Long, and B.A. Sullenger. 2003. Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes. RNA 9, 1254-1263 

  2. Honda, M., M.R. Beard, L.H. Ping, and S.M. Lemon. 1999. A phylogenetically conserved stem-loop structure at the 5' border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation. J. Virol. 73, 1165-1174 

  3. Hugle, T. and A. Cerny. 2003. Current therapy and new molecular approaches to antiviral treatment and prevention of hepatitis C. Rev. Med. Virol. 13, 361-371 

  4. Jones, J.T., S.-W. Lee, and B.A. Sullenger. 1996. Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nat. Med. 2, 643-648 

  5. Kohler, U., B.G. Ayre, H.M. Goodman, and J. Haseloff. 1999. Trans-splicing ribozymes for targeted gene delivery. J. Mol. Biol. 185, 1935-1950 

  6. Lan, N., R.P. Howrey, S.-W. Lee, C.A. Smith, and B.A. Sullenger. 1998. Ribozyme-mediated repair of sickle $\beta$ -globin mRNAs in erythrocyte precursors. Science 280, 1593-1596. 

  7. Lauer, G.M. and B.D. Waker. 2001. Hepatitis C virus infection. N. Engl. J. Med. 345, 41-52 

  8. Park Y.-H., H.-S. Jung, B.-S. Kwon, and S.-W. Lee. 2003. Replacement of thymidine phosphorylase RNA with group I intron of Tetrahymena thermophila by targeted trans-splicing. J. Microbiol. 41, 340-344 

  9. Phylactou, L.A., C. Darrah, and M.A.J. Wood. 1998. Ribozymemediated trans-splicing of a trinucleotide repeat. Nat. Genet. 18, 378-381 

  10. Rogers, C.S., C.G. Vanoye, B.A. Sullenger and A.L. George, Jr. 2002. Functional repair of a mutant chloride channel using a trans-splicing ribozyme. J. Clin. Invest. 110, 1783-1798 

  11. Rosenberg, S. 2001. Recent advances in the molecular biology of hepatitis C virus. J. Mol. Biol. 313, 451-464 

  12. Ryu, K.-J. and S.-W. Lee. 2003. Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site. J. Biochem. Mol. Biol. 36, 538-544 

  13. Ryu, K.-J., J.-H. Kim, and S.-W. Lee. 2003. Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted transsplicing. Mol. Ther. 7, 386-395 

  14. Shin, K.-S., B.A. Sullenger, and S.-W. Lee. 2004. Ribozyme-mediated induction of apoptosis in human cancer cells by targeted repair of mutant p53 RNA. Mol. Ther. 10, 365-372 

  15. Watanabe, T. and B.A. Sullenger. 2000. Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc. Natl. Acad. Sci. USA 97, 8490-8494 

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