Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Feb 15;25(4):769–775. doi: 10.1093/nar/25.4.769

The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells.

R Hormes 1, M Homann 1, I Oelze 1, P Marschall 1, M Tabler 1, F Eckstein 1, G Sczakiel 1
PMCID: PMC146489  PMID: 9016627

Abstract

The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-1tat-directed asymmetric hammerhead ribozyme alphaYRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of successively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for efficient association with a 645 nt target RNA transcript in vitro(for all constructs kass ranged between 0.3 and 1.8x104/M/s). The cleavage rate constants (kcleav) were independent of the length of the antisense flank and ranged between 0.8 and 1.2x10-4/s. However, the length of the antisense arms, as well as the mode of delivery and the subcellular location of the ribozymes, had a dramatic effect on efficacy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozymes were co-microinjected into the nucleus of human cells, a minimum length of 51 nt in the antisense arm was necessary for antisense- and ribozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorter antisense arms were almost ineffective. Conversely, short chain ribozymes, including those with chemical modifications, were superior to long chain ribozymes when co-microinjected into the cytoplasm. When transfected, all ribozymes showed an antisense effect as well as an additional ribozyme-mediated increase in inhibition. Consequences for the design and application of ribozymes are discussed.

Full Text

The Full Text of this article is available as a PDF (162.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beck J., Nassal M. Efficient hammerhead ribozyme-mediated cleavage of the structured hepatitis B virus encapsidation signal in vitro and in cell extracts, but not in intact cells. Nucleic Acids Res. 1995 Dec 25;23(24):4954–4962. doi: 10.1093/nar/23.24.4954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beigelman L., McSwiggen J. A., Draper K. G., Gonzalez C., Jensen K., Karpeisky A. M., Modak A. S., Matulic-Adamic J., DiRenzo A. B., Haeberli P. Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance. J Biol Chem. 1995 Oct 27;270(43):25702–25708. doi: 10.1074/jbc.270.43.25702. [DOI] [PubMed] [Google Scholar]
  4. Bertrand E. L., Rossi J. J. Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1. EMBO J. 1994 Jun 15;13(12):2904–2912. doi: 10.1002/j.1460-2075.1994.tb06585.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bertrand E., Pictet R., Grange T. Can hammerhead ribozymes be efficient tools to inactivate gene function? Nucleic Acids Res. 1994 Feb 11;22(3):293–300. doi: 10.1093/nar/22.3.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bratty J., Chartrand P., Ferbeyre G., Cedergren R. The hammerhead RNA domain, a model ribozyme. Biochim Biophys Acta. 1993 Dec 14;1216(3):345–359. doi: 10.1016/0167-4781(93)90001-t. [DOI] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crisell P., Thompson S., James W. Inhibition of HIV-1 replication by ribozymes that show poor activity in vitro. Nucleic Acids Res. 1993 Nov 11;21(22):5251–5255. doi: 10.1093/nar/21.22.5251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Flory C. M., Pavco P. A., Jarvis T. C., Lesch M. E., Wincott F. E., Beigelman L., Hunt S. W., 3rd, Schrier D. J. Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint. Proc Natl Acad Sci U S A. 1996 Jan 23;93(2):754–758. doi: 10.1073/pnas.93.2.754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  11. Heidenreich O., Benseler F., Fahrenholz A., Eckstein F. High activity and stability of hammerhead ribozymes containing 2'-modified pyrimidine nucleosides and phosphorothioates. J Biol Chem. 1994 Jan 21;269(3):2131–2138. [PubMed] [Google Scholar]
  12. Heidenreich O., Kang S. H., Brown D. A., Xu X., Swiderski P., Rossi J. J., Eckstein F., Nerenberg M. Ribozyme-mediated RNA degradation in nuclei suspension. Nucleic Acids Res. 1995 Jun 25;23(12):2223–2228. doi: 10.1093/nar/23.12.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Heidenreich O., Pieken W., Eckstein F. Chemically modified RNA: approaches and applications. FASEB J. 1993 Jan;7(1):90–96. doi: 10.1096/fasebj.7.1.7678566. [DOI] [PubMed] [Google Scholar]
  14. Herschlag D. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn't always better. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):6921–6925. doi: 10.1073/pnas.88.16.6921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hertel K. J., Herschlag D., Uhlenbeck O. C. A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry. 1994 Mar 22;33(11):3374–3385. doi: 10.1021/bi00177a031. [DOI] [PubMed] [Google Scholar]
  16. Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Homann M., Nedbal W., Sczakiel G. Dissociation of long-chain duplex RNA can occur via strand displacement in vitro: biological implications. Nucleic Acids Res. 1996 Nov 15;24(22):4395–4400. doi: 10.1093/nar/24.22.4395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Homann M., Rittner K., Sczakiel G. Complementary large loops determine the rate of RNA duplex formation in vitro in the case of an effective antisense RNA directed against the human immunodeficiency virus type 1. J Mol Biol. 1993 Sep 5;233(1):7–15. doi: 10.1006/jmbi.1993.1480. [DOI] [PubMed] [Google Scholar]
  19. Homann M., Tzortzakaki S., Rittner K., Sczakiel G., Tabler M. Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucleic Acids Res. 1993 Jun 25;21(12):2809–2814. doi: 10.1093/nar/21.12.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. James W., Crisell P., Rhodes A. Gene inhibition of HIV-1 replication. A comparative and mechanistic study. Ann N Y Acad Sci. 1992 Oct 28;660:274–275. doi: 10.1111/j.1749-6632.1992.tb21082.x. [DOI] [PubMed] [Google Scholar]
  21. James W., al-Shamkhani A. RNA enzymes as tools for gene ablation. Curr Opin Biotechnol. 1995 Feb;6(1):44–49. doi: 10.1016/0958-1669(95)80008-5. [DOI] [PubMed] [Google Scholar]
  22. Kumar A., Wilson S. H. Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 1990 Dec 4;29(48):10717–10722. doi: 10.1021/bi00500a001. [DOI] [PubMed] [Google Scholar]
  23. Lyngstadaas S. P., Risnes S., Sproat B. S., Thrane P. S., Prydz H. P. A synthetic, chemically modified ribozyme eliminates amelogenin, the major translation product in developing mouse enamel in vivo. EMBO J. 1995 Nov 1;14(21):5224–5229. doi: 10.1002/j.1460-2075.1995.tb00207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oberosler P., Hloch P., Ramsperger U., Stahl H. p53-catalyzed annealing of complementary single-stranded nucleic acids. EMBO J. 1993 Jun;12(6):2389–2396. doi: 10.1002/j.1460-2075.1993.tb05893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pachuk C. J., Yoon K., Moelling K., Coney L. R. Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences. Nucleic Acids Res. 1994 Feb 11;22(3):301–307. doi: 10.1093/nar/22.3.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Perriman R., Delves A., Gerlach W. L. Extended target-site specificity for a hammerhead ribozyme. Gene. 1992 Apr 15;113(2):157–163. doi: 10.1016/0378-1119(92)90391-2. [DOI] [PubMed] [Google Scholar]
  27. Pontius B. W., Berg P. Rapid renaturation of complementary DNA strands mediated by cationic detergents: a role for high-probability binding domains in enhancing the kinetics of molecular assembly processes. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8237–8241. doi: 10.1073/pnas.88.18.8237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pontius B. W., Berg P. Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8403–8407. doi: 10.1073/pnas.87.21.8403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Portman D. S., Dreyfuss G. RNA annealing activities in HeLa nuclei. EMBO J. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  31. Rittner K., Burmester C., Sczakiel G. In vitro selection of fast-hybridizing and effective antisense RNAs directed against the human immunodeficiency virus type 1. Nucleic Acids Res. 1993 Mar 25;21(6):1381–1387. doi: 10.1093/nar/21.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
  33. Sczakiel G., Goody R. S. Antisense principle or ribozyme action? Biol Chem Hoppe Seyler. 1994 Nov;375(11):745–746. [PubMed] [Google Scholar]
  34. Sczakiel G., Nedbal W. The potential of ribozymes as antiviral agents. Trends Microbiol. 1995 Jun;3(6):213–217. doi: 10.1016/s0966-842x(00)88927-1. [DOI] [PubMed] [Google Scholar]
  35. Sczakiel G., Pawlita M., Kleinheinz A. Specific inhibition of human immunodeficiency virus type 1 replication by RNA transcribed in sense and antisense orientation from the 5'-leader/gag region. Biochem Biophys Res Commun. 1990 Jun 15;169(2):643–651. doi: 10.1016/0006-291x(90)90379-2. [DOI] [PubMed] [Google Scholar]
  36. Shimayama T., Nishikawa S., Taira K. Extraordinary enhancement of the cleavage activity of a DNA-armed hammerhead ribozyme at elevated concentrations of Mg2+ ions. FEBS Lett. 1995 Jul 17;368(2):304–306. doi: 10.1016/0014-5793(95)00682-y. [DOI] [PubMed] [Google Scholar]
  37. Shimayama T., Nishikawa S., Taira K. Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. Biochemistry. 1995 Mar 21;34(11):3649–3654. doi: 10.1021/bi00011a020. [DOI] [PubMed] [Google Scholar]
  38. Symons R. H. Small catalytic RNAs. Annu Rev Biochem. 1992;61:641–671. doi: 10.1146/annurev.bi.61.070192.003233. [DOI] [PubMed] [Google Scholar]
  39. Tabler M., Homann M., Tzortzakaki S., Sczakiel G. A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design. Nucleic Acids Res. 1994 Sep 25;22(19):3958–3965. doi: 10.1093/nar/22.19.3958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsuchihashi Z., Khosla M., Herschlag D. Protein enhancement of hammerhead ribozyme catalysis. Science. 1993 Oct 1;262(5130):99–102. doi: 10.1126/science.7692597. [DOI] [PubMed] [Google Scholar]
  41. Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
  42. Wu L., Bayle J. H., Elenbaas B., Pavletich N. P., Levine A. J. Alternatively spliced forms in the carboxy-terminal domain of the p53 protein regulate its ability to promote annealing of complementary single strands of nucleic acids. Mol Cell Biol. 1995 Jan;15(1):497–504. doi: 10.1128/mcb.15.1.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zoumadakis M., Tabler M. Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes. Nucleic Acids Res. 1995 Apr 11;23(7):1192–1196. doi: 10.1093/nar/23.7.1192. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES