PCR-Based Construction of Long Hammerhead Ribozymes

  • Martin Zillmann
  • Gregory Robinson
Part of the Methods in Molecular Biology™ book series (MIMB, volume 74)


With the advent of antisense technology, there has been much interest in the use of long RNAs expressed in vivo to inhibit the expression of target genes. More recently, there have been numerous reports that the incorporation of either hairpin or hammerhead ribozyme motifs (catalytic antisense) into such RNAs increases their effectiveness (1, 2, 3). This section will describe a generally applicable, simple, PCR-based method to construct catalytic antisense RNA containing the hammerhead catalytic core that will cleave the target of interest at a GUC sequence. All discussion will focus on the hammerhead motif, although, in principle, the hairpin motif could be incorporated instead.


Microcentrifuge Tube Hammerhead Ribozyme Hairpin Motif Final Insert Incomplete Denaturation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    De Young, M. B., Kincade-Denker, J., Boehm, C. A., Riek, R. P., Mamone, J. A., McSwiggen, J. A., and Graham, R. M. (1994) Functional characterization of ribozymes expressed using U1 and T7 vectors for the intracellular cleavage of ANF mRNA. Biochemistry 33, 12,127–12,138.PubMedCrossRefGoogle Scholar
  2. 2.
    Homann, M., Tabler, M., Tzortzakaki, S., and Sczakiel, G. (1994) Extension of helix II of an HIV-1-directed hammerhead ribozyme with long antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in having cells. Nucleic Acids Res. 22, 3951–3957.PubMedCrossRefGoogle Scholar
  3. 3.
    Lieber, A. and Strauss, M. (1995) Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell Biol. 15, 540–551.PubMedGoogle Scholar
  4. 4.
    Cantor, G. H., McElwain, T. F., Birkebak, T. A., and Palmer, G. H. (1993) Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression. Proc. Natl. Acad. Sci. USA 90, 10,932–10,936.PubMedCrossRefGoogle Scholar
  5. 5.
    Deshler, J. O., Li, H., Rossi, J. J., and Castanotto, D. (1995) Ribozymes expressed within the loop of a natural antisense RNA form functional transcription terminators. Gene 155, 35–43.PubMedCrossRefGoogle Scholar
  6. 6.
    Ellis, J. and Rogers, J. (1993) Design and specificity of hammerhead ribozymes against calretinin mRNA. Nucleic Acids Res. 21, 5171–5178.PubMedCrossRefGoogle Scholar
  7. 7.
    Ferbeyre, G., Bratty, J., Chen, H., and Cedergren, R. (1995) A hammerhead ribozyme inhibits ADE1_gene expression in yeast. Gene 155, 45–50.PubMedCrossRefGoogle Scholar
  8. 8.
    Gast, F.-U., Amiri, K. M. A., and Hagerman, P. J. (1994) Interhelix geometry of stems I and II of a self-cleaving hammerhead RNA. Biochemistry 35, 1788–1796.CrossRefGoogle Scholar
  9. 9.
    Mazzolini, L., Axelos, M., Lescure, N., and Yot, P. (1992) Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts. Plant Mol. Biol. 20, 715–731.PubMedCrossRefGoogle Scholar
  10. 10.
    Crisell, P., Thompson, S., and James, W. (1993) Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro. Nucleic Acids Res. 21, 5251–5255.PubMedCrossRefGoogle Scholar
  11. 11.
    Kobayashi, H., Dorai, T., Holland, J. F., and Olinuma, T. (1994) Reversal of drug sensitivity in multidrug-resistant tumor cells by MDR1 (PGY1) ribozyme. Cancer Res 54, 1271–1275.PubMedGoogle Scholar
  12. 12.
    Sane, D. C. and Chema, D. J. (1995) PCR-based production of a ribozyme to plasminogen activator inhibitor I. Biotechniques 18, 208–210.PubMedGoogle Scholar
  13. 13.
    Tabler, M., Homann, M., Tzortzakaki, S., and Sczakiel, G. (1994) A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme advantages of an asymmetric design. Nucleic Acids Res. 22, 3958–3965.PubMedCrossRefGoogle Scholar
  14. 14.
    Higuchi, R., Krummel, B., and Saiko, R. K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367.PubMedCrossRefGoogle Scholar
  15. 15.
    Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Ehrlich, H. (1986) Specific enzymatic amplification of DNA in vitro the polymerase chain reaction. Cold Spring Harbor Sym. 51, 263–273.Google Scholar
  16. 16.
    Vallette, F., Mege, E., Reiss, A., and Adesnik, M. (1989) Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res. 17, 723–733.PubMedCrossRefGoogle Scholar
  17. 17.
    Noonberg, S. B., Scott, G. K., Garovoy, M. R., Benz, C. C., and Hunt, C. A. (1994) In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation. Nucleic Acids Res. 22, 2830–2836.PubMedCrossRefGoogle Scholar
  18. 18.
    Fedor, M. and Uhlenbeck, O. (1990) Substrate sequence effects of “hammerhead” RNA catalytic efficiency. Proc. Natl. Acad. Sci. USA 87, 1668–1672.PubMedCrossRefGoogle Scholar
  19. 19.
    Woodson, S. and Cech, T. (1991) Alternative secondary structures in the 5′ exon affects both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA. Biochemistry 30, 2042–2050.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1997

Authors and Affiliations

  • Martin Zillmann
    • 1
  • Gregory Robinson
    • 1
  1. 1.HybridonWorcester

Personalised recommendations