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Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology

Nature Biotechnology volume 19, pages 56–61 (2001)Cite this article

Abstract

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

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Figure 1: Principle of the assay and primary fluorescence measurements with HHR1/SL1.
Figure 2: 32P-Labeling experiments under single- and multiple-turnover conditions, Ki determinations and cooperativity.
Figure 3: Statistical analysis of assay accuracy.
Figure 4: In vivo cleavage activity of the cis-snorbozyme in the presence and absence of TubA.

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References

  1. Gait, M.J. & Karn, J. RNA recognition by the human immunodeficiency virus Tat and Rev proteins. Trends Biochem. Sci. 18, 255–259 ( 1993).

    Article  CAS  Google Scholar 

  2. Hermann, T. & Westhof, E. RNA as a drug target: chemical, modelling, and evolutionary tools. Curr. Opin. Biotechnol. 9, 66–73 (1998 ).

    Article  CAS  Google Scholar 

  3. Afshar, M., Prescott, C.D. & Varani, G. Structure-based and combinatorial search for new RNA-binding drugs. Curr. Opin. Biotechnol. 10, 59– 63 (1999).

    Article  CAS  Google Scholar 

  4. Ecker, D.J. & Griffey, R.H. RNA as a small-molecule drug target: doubling the value of genomics. Drug Discovery Today 4, 420–429 ( 1999).

    Article  CAS  Google Scholar 

  5. Woodcock, J., Moazed, D., Cannon, M., Davies, J. & Noller, H.F. Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA. EMBO J. 10, 3099–3103 (1991).

    Article  CAS  Google Scholar 

  6. Zapp, M.L., Stern, S. & Green, M.R. Small molecules that selectively block RNA binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell 74, 969–978 ( 1993).

    Article  CAS  Google Scholar 

  7. Wang, S., Huber, P.W., Cui, M., Czarnik, A.W. & Mei, H.Y. Binding of neomycin to the TAR element of HIV-1 RNA induces dissociation of Tat protein by an allosteric mechanism . Biochemistry 37, 5549– 5557 (1998).

    Article  CAS  Google Scholar 

  8. Wang, Y., Hamasaki, K. & Rando, R.R. Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region . Biochemistry 36, 768– 779 (1997).

    Article  CAS  Google Scholar 

  9. Walter, F., Vicens, Q. & Westhof, E. Aminoglycoside–RNA interactions. Curr. Opin. Chem. Biol. 3, 694–704 (1999).

    Article  CAS  Google Scholar 

  10. Schroeder, R., Waldsich, C. & Wank, H. Modulation of RNA function by aminoglycoside antibiotics. EMBO J. 19, 1– 9 (2000).

    Article  CAS  Google Scholar 

  11. Hermann, T. & Westhof, E. Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA. J. Mol. Biol. 276, 903–912 (1998).

    Article  CAS  Google Scholar 

  12. Murray, J.B. & Arnold, J.R. Antibiotic interactions with the hammerhead ribozyme: tetracyclines as a new class of hammerhead inhibitor . Biochem. J. 317, 855– 860 (1996).

    Article  CAS  Google Scholar 

  13. Clouet-d'Orval, B., Stage, T.K. & Uhlenbeck, O.C. Neomycin inhibition of the hammerhead ribozyme involves ionic interactions. Biochemistry 34, 11186–11190 (1995).

    Article  CAS  Google Scholar 

  14. Stage, T.K., Hertel, K.J. & Uhlenbeck, O.C. Inhibition of the hammerhead ribozyme by neomycin . RNA 1, 95–101 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Michael, K. & Tor, Y. Designing novel RNA Binders . Chemistry Eur. J. 4, 2091– 2098 (1998).

    Article  CAS  Google Scholar 

  16. Scott, W.G., Murray, J.B., Arnold, J.R.P., Stoddard, B.L. & Klug, A. Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 274, 2065–2069 ( 1996).

    Article  CAS  Google Scholar 

  17. Pearson, N.D. & Prescott, C.D. RNA as a drug target . Chem. Biol. 4, 409–414 (1997).

    Article  CAS  Google Scholar 

  18. Stage-Zimmermann, T.K. & Uhlenbeck, O.C. Hammerhead ribozyme kinetics. RNA 4, 875– 889 (1998).

    Article  CAS  Google Scholar 

  19. Tor, Y. RNA and the small molecule world. Angew. Chem. Int. Edn. 38, 1579–1582 (1999).

    Article  CAS  Google Scholar 

  20. Noller, H.F. Ribosomes. Drugs and the RNA world. Nature 353, 302–303 (1991).

    Article  CAS  Google Scholar 

  21. Rogers, J., Chang, A.H., von Ahsen, U., Schroeder, R. & Davies, J. Inhibition of the self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics. J. Mol. Biol. 259, 916–925 (1996).

    Article  CAS  Google Scholar 

  22. Neu, H.C. The crisis in antibiotic resistance. Science 257, 1064–1073 (1992).

    Article  CAS  Google Scholar 

  23. Davies, J. & Wright, G.D. Bacterial resistance to aminoglycoside antibiotics. Trends Microbiol. 5, 234–240 (1997).

    Article  CAS  Google Scholar 

  24. Davies, J. Inactivation of antibiotics and the dissemination of resistance genes. Science 264, 375–382 ( 1994).

    Article  CAS  Google Scholar 

  25. Jenne, A., Gmelin, W., Raffler, N. & Famulok, M. Real-time characterization of ribozymes by fluorecence resonance energy transfer (FRET). Angew. Chem. Int. Edn. 38, 1300–1303 (1999).

    Article  CAS  Google Scholar 

  26. Singh, K.K., Parwaresch, R. & Krupp, G. Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET). RNA 5, 1348–1356 ( 1999).

    Article  CAS  Google Scholar 

  27. von Ahsen, U., Davies, J. & Schroeder, R. Non-competitive inhibition of group I intron RNA self-splicing by aminoglycoside antibiotics. J. Mol. Biol. 226, 935–941 ( 1992).

    Article  CAS  Google Scholar 

  28. Hoch, I., Berens, C., Westhof, E. & Schroeder, R. Antibiotic inhibition of RNA catalysis: neomycin B binds to the catalytic core of the td group I intron displacing essential metal ions. J. Mol. Biol. 282, 557–569 ( 1998).

    Article  CAS  Google Scholar 

  29. Mikkelsen, N.E., Brannvall, M., Virtanen, A. & Kirsebom, L.A. Inhibition of RNase P RNA cleavage by aminoglycosides . Proc. Natl. Acad. Sci. USA 96, 6155– 6160 (1999).

    Article  CAS  Google Scholar 

  30. Earnshaw, D.J. & Gait, M.J. Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions. Nucleic Acids Res. 26 , 5551–5561 (1998).

    Article  CAS  Google Scholar 

  31. Zhu, K., Henning, D., Iwakuma, T., Valdez, B.C. & Busch, H. Adriamycin inhibits human RH II/Gu RNA helicase activity by binding to its substrate. Biochem. Biophys. Res. Commun. 266, 361–365 (1999).

    Article  CAS  Google Scholar 

  32. Dassonneville, L., Hamy, F., Colson, P., Houssier, C. & Bailly, C. Binding of Hoechst 33258 to the TAR RNA of HIV-1. Recognition of a pyrimidine bulge-dependent structure . Nucleic Acids Res. 25, 4487– 4492 (1997).

    Article  CAS  Google Scholar 

  33. Mestre, B. et al. Oligonucleotide inhibition of the interaction of HIV-1 Tat protein with the trans-activation responsive region (TAR) of HIV RNA. Biochim. Biophys. Acta 1445, 86–98 (1999).

    Article  CAS  Google Scholar 

  34. Werstuck, G. & Green, M.R. Controlling gene expression in living cells through small molecule–RNA interactions. Science 282, 296–298 ( 1998).

    Article  CAS  Google Scholar 

  35. Bailly, C., Colson, P., Houssier, C. & Hamy, F. The binding mode of drugs to the TAR RNA of HIV-1 studied by electric linear dichroism. Nucleic Acids Res. 24, 1460– 1464 (1996).

    Article  CAS  Google Scholar 

  36. Pilch, D.S., Kirolos, M.A., Liu, X., Plum, G.E. & Breslauer, K.J. Berenil [1,3-bis(4′-amidinophenyl)triazene] binding to DNA duplexes and to a RNA duplex: evidence for both intercalative and minor groove binding properties. Biochemistry 34 , 9962–9976 (1995).

    Article  CAS  Google Scholar 

  37. Samarsky, D.A. et al. A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency. Proc. Natl. Acad. Sci. USA 96, 6609–6614 ( 1999).

    Article  CAS  Google Scholar 

  38. Rossi, J.J. Ribozymes in the nucleolus. Science 285, 1685 (1999).

    Article  CAS  Google Scholar 

  39. Samarsky, D.A., Fournier, M.J., Singer, R.H. & Bertrand, E. The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization. EMBO J. 17, 3747–3757 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Barbara Waters and TerraGen Discovery for providing the actinomycete extracts and Abbott Laboratories for chelocardin, E. Bertrand, E. Westhof, and the members of the Famulok laboratory for helpful discussions, and M.J. Fournier for sharing the strategic approach of the in vivo cleavage assay. This work was supported by the Deutsche Forschungsgemeinschaft, Aventis GenCell (to M.F. and N.P.), and the H. Arthur Smith Foundation for Cancer Research (to D.A.S.).

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Author notes

  1. Andreas Jenne

    Present address: NascaCell GmbH, Eisvogelweg 2, D-83209, Prien am Chiemsee, Germany

  2. Nicolas Piganeau

    Present address: Aventis Gencell, 3825 Bay Center Place, Hayward, CA, 94545, USA

Authors and Affiliations

  1. Kekulé-Institut für Organische Chemie und Biochemie, Gerhard-Domagk-Str. 1, Bonn, 35121, Germany

    Andreas Jenne, Jörg S. Hartig, Nicolas Piganeau & Michael Famulok

  2. Institut für Biochemie, LMU München, Feodor-Lynen-Str. 25, München, 81377, Germany

    Andreas Tauer

  3. Howard Hughes Medical Institute, Programs in Gene Function and Expression and Molecular Medicine, University of Massachusetts Medical School, 373 Plantation St., Worcester, 01605, MA, USA

    Dmitry A. Samarsky & Michael R. Green

  4. Department of Microbiology and Immunology, University of British Columbia, Vancouver, V6T 1Z3 , BC, Canada

    Julian Davies

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Correspondence to Michael Famulok.

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Jenne, A., Hartig, J., Piganeau, N. et al. Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology. Nat Biotechnol 19, 56–61 (2001). https://doi.org/10.1038/83513

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