Abstract
Triplex ribozymes allow for the individual activity of multiple trans-acting ribozymes producing higher target cleavage relative to tandem-expressed RZs. A triplex expression system based on a single hairpin ribozyme for the multiple expression (multiplex) vectors can be engineered to target RNAs with single or multiple antisense-accessible sites. System construction relies on triplex expression modules consisting of hairpin ribozyme cassettes flanked by ribozymes lacking catalytic domains. Multiplex vectors can be generated with single or multiple specificity by tandem cloning of triplex expression modules. Triplex ribozymes are initially tested in vitro using cis- and trans-cleavage assays against radioactive-labeled targets. In addition, triplex ribozymes are tested for cis and trans cleavage in vivo by transfection in cultured cells followed by ribonuclease protection assays (RPAs) and RT-PCR. The use of triplex configurations with multiplex ribozymes will provide the basis for the development of future RZ-based therapies and technologies.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Benitez-Hess, M.L. and Alvarez-Salas, L.M. (2006) Utilization of ribozymes as antiviral agents. Lett Drug Des Discov, 3, 390–404.
Burke, J.M. (2002) Hairpin and hammerhead ribozymes: how different are they? Biochem Soc Trans, 30, 1115–1118.
Haseloff, J. and Gerlach, W.L. (1989) Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus. Gene, 82, 43–52.
Hampel, A. (1998) The hairpin ribozyme: discovery, two-dimensional model, and development for gene therapy. Prog Nucleic Acid Res Mol Biol, 58, 1–39.
Berzal-Herranz, A., Joseph, S., Chowrira, B.M., Butcher, S.E., and Burke, J.M. (1993) Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme. EMBO J, 12, 2567–2573.
Walter, N.G. and Burke, J.M. (1998) The hairpin ribozyme: structure, assembly and catalysis. Curr Opin Chem Biol, 2, 24–30.
Berzal-Herranz, A. and Burke, J.M. (1997) Ligation of RNA molecules by the hairpin ribozyme. Methods Mol Biol, 74, 349–355.
Shin, C., Choi, J.N., Song, S.I., Song, J.T., Ahn, J.H., Lee, J.S., and Choi, Y.D. (1996) The loop B domain is physically separable from the loop A domain in the hairpin ribozyme. Nucleic Acids Res, 24, 2685–2689.
Taylor, N.R. and Rossi, J.J. (1991) Ribozyme-mediated cleavage of an HIV-1 gag RNA: the effects of nontargeted sequences and secondary structure on ribozyme cleavage activity. Antisense Res Dev, 1, 173–186.
Alvarez-Salas, L.M., Cullinan, A.E., Siwkowski, A., Hampel, A., and DiPaolo, J.A. (1998) Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes. Proc Natl Acad Sci U S A, 95, 1189–1194.
Michienzi, A. and Rossi, J.J. (2001) Intracellular applications of ribozymes. Methods Enzymol, 341, 581–596.
Taira, K., Oda, M., Shinshi, H., Maeda, H., and Furukawa, K. (1990) Construction of a novel artificial-ribozyme-releasing plasmid. Protein Eng, 3, 733–737.
Altschuler, M., Tritz, R., and Hampel, A. (1992) A method for generating transcripts with defined 5' and 3' termini by autolytic processing. Gene, 122, 85–90.
Ohkawa, J., Yuyama, N., Takebe, Y., Nishikawa, S., and Taira, K. (1993) Importance of independence in ribozyme reactions: kinetic behavior of trimmed and of simply connected multiple ribozymes with potential activity against human immunodeficiency virus. Proc Natl Acad Sci U S A, 90, 11302–11306.
Aquino-Jarquin, G., Benitez-Hess, M.L., DiPaolo, J.A., and Alvarez-Salas, L.M. (2008) A triplex ribozyme expression system based on a single hairpin ribozyme. Oligonucleotides, 18, 213–224.
Benitez-Hess, M.L., DiPaolo, J.A., and Alvarez-Salas, L.M. (2004) Antisense activity detection by inhibition of fluorescence resonance energy transfer. Luminescence, 19, 85–93.
Guapillo, M.R., Marquez-Gutiérrez, M.A., Benitez-Hess, M.L., and Alvarez-Salas, L.M. (2006) A bacterial reporter system for the evaluation of antisense oligodeoxynucleotides directed against human papillomavirus type 16 (HPV-16). Arch Med Res, 37, 584–592.
DeYoung, M.B., Siwkowski, A., and Hampel, A. (1997) Determination of catalytic parameters for hairpin ribozymes. Methods Mol Biol, 74, 209–220.
Marquez-Gutierrez, M.A., Benitez-Hess, M.L., DiPaolo, J.A., and Alvarez-Salas, L.M. (2007) Effect of combined antisense oligodeoxynucleotides directed against the human papillomavirus type 16 on cervical carcinoma cells. Arch Med Res, 38, 730–738.
Acknowledgments
The authors would like to thank Dr. Joseph A. DiPaolo (NCI/NIH) for his unconditional support and encouragement. We also thank Dr. María Luisa Benítez-Hess for excellent technical assistance. This project was partially supported by CONACyT (Grant No. 45715Z).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Aquino-Jarquin, G., Rojas-Hernández, R., Alvarez-Salas, L.M. (2010). Design and Function of Triplex Hairpin Ribozymes. In: Sioud, M. (eds) RNA Therapeutics. Methods in Molecular Biology, vol 629. Humana Press. https://doi.org/10.1007/978-1-60761-657-3_21
Download citation
DOI: https://doi.org/10.1007/978-1-60761-657-3_21
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-656-6
Online ISBN: 978-1-60761-657-3
eBook Packages: Springer Protocols