Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Protein-dependent ribozymes report molecular interactions in real time

Nature Biotechnology volume 20, pages 717–722 (2002)Cite this article

Abstract

Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein–protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Rev-responsive and aptamer-inhibited ribozymes.
Figure 2: Interaction of Rev with small molecules.
Figure 3: Coumermycin A1 inhibits HIV-1 replication.
Figure 4: Specific protein–protein interactions as a function of cleavage activities for AHH-Thr and AHP-Thr.

Similar content being viewed by others

References

  1. Thompson, L. & Ellman, J. Synthesis and applications of small molecule libraries. Chem. Rev. 96, 555–600 (1996).

    Article  CAS  Google Scholar 

  2. Jayawickreme, C.K. & Kost, T.A. Gene expression systems in the development of high-throughput screens. Curr. Opin. Biotechnol. 8, 629–634 (1997).

    Article  CAS  Google Scholar 

  3. Rademann, J. & Jung, G. Drug discovery. Integrating combinatorial synthesis and bioassays. Science 287, 1947–1948 (1998).

    Article  Google Scholar 

  4. Mendelsohn, A.R. & Brent, R. Protein interaction methods—toward an endgame. Science 284, 1948–1950 (1999).

    Article  CAS  Google Scholar 

  5. Pandey, A. & Mann, M. Proteomics to study genes and genomes. Nature 405, 837–846 (2000).

    Article  CAS  Google Scholar 

  6. Phizicky, E.M. & Fields, S. Protein–protein interactions: methods for detection and analysis. Microbiol. Rev. 59, 94–123 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Cech, T.R. in The RNA World (eds Gesteland, R.F. & Atkins, J.F.) 239–269 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1993).

    Google Scholar 

  8. Warashina, M., Takagi, Y., Stec, W.J. & Taira, K. Differences among mechanisms of ribozyme-catalyzed reactions. Curr. Opin. Biotechnol. 11, 354–362 (2000).

    Article  CAS  Google Scholar 

  9. Long, D.M. & Uhlenbeck, O.C. Self-cleaving catalytic RNA. FASEB J. 7, 25–30 (1993).

    Article  CAS  Google Scholar 

  10. Doherty, E.A. & Doudna, J.A. Ribozyme structures and mechanisms. Annu. Rev. Biochem. 69, 597–615 (2000).

    Article  CAS  Google Scholar 

  11. Walter, N.G. & Burke, J.M. The hairpin ribozyme: structure, assembly and catalysis. Curr. Opin. Chem. Biol. 2, 24–30 (1998).

    Article  CAS  Google Scholar 

  12. Verma, S., Vaish, N.K. & Eckstein, F. Structure–function studies of the hammerhead ribozyme. Curr. Opin. Chem. Biol. 1, 532–536 (1997).

    Article  CAS  Google Scholar 

  13. Rossi, J.J. Ribozymes, genomics and therapeutics. Chem. Biol. 6, R33–R37 (1999).

    Article  CAS  Google Scholar 

  14. Bramlage, B., Luzi, E. & Eckstein, F. Designing ribozymes for the inhibition of gene expression. Trends Biotechnol. 16, 434–438 (1998).

    Article  CAS  Google Scholar 

  15. Sullenger, B.A. & Cech, T.R. Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing. Nature 371, 619–622 (1994).

    Article  CAS  Google Scholar 

  16. Sullenger, B.A. Revising messages traveling along the cellular information superhighway. Chem. Biol. 2, 249–253 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Jenne, A. et al. Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology. Nat. Biotechnol. 19, 56–61 (2001).

    Article  CAS  Google Scholar 

  19. Soukup, G.A. & Breaker, R.R. Engineering precision RNA molecular switches. Proc. Natl. Acad. Sci. USA 96, 3584–3589 (1999).

    Article  CAS  Google Scholar 

  20. Peterson, R.D. & Feigon, J. Structural change in Rev responsive element RNA of HIV-1 on binding Rev peptide. J. Mol. Biol. 264, 863–877 (1996).

    Article  CAS  Google Scholar 

  21. Gosser, Y. et al. Peptide-triggered conformational switch in HIV-1 RRE RNA complexes. Nat. Struct. Biol. 8, 146–150 (2001).

    Article  CAS  Google Scholar 

  22. Giver, L. et al. Selective optimization of the Rev-binding element of HIV-1. Nucleic Acids Res. 21, 5509–5516 (1993).

    Article  CAS  Google Scholar 

  23. Battiste, J.L. et al. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science 273, 1547–1551 (1996).

    Article  CAS  Google Scholar 

  24. Hung, L.W., Holbrook, E.L. & Holbrook, S.R. The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability. Proc. Natl. Acad. Sci. USA 97, 5107–5112 (2000).

    Article  CAS  Google Scholar 

  25. Ippolito, J.A. & Steitz, T.A. The structure of the HIV-1 RRE high affinity rev binding site at 1.6 Å resolution. J. Mol. Biol. 295, 711–717 (2000).

    Article  CAS  Google Scholar 

  26. Bohan, C.A. et al. Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro. Gene Expr. 2, 391–407 (1992).

    CAS  PubMed  Google Scholar 

  27. Burd, C.G. & Dreyfuss, G. Conserved structures and diversity of functions of RNA-binding proteins. Science 265, 615–621 (1994).

    Article  CAS  Google Scholar 

  28. Mattaj, I.W. RNA recognition: a family matter? Cell 73, 837–840 (1993).

    Article  CAS  Google Scholar 

  29. Patel, D.J. Adaptive recognition in RNA complexes with peptides and protein modules. Curr. Opin. Struct. Biol. 9, 74–87 (1999).

    Article  CAS  Google Scholar 

  30. Esteban, J.A., Banerjee, A.R. & Burke, J.M. Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations. J. Biol. Chem. 272, 13629–13639 (1997).

    Article  CAS  Google Scholar 

  31. Bock, L.C., Griffin, L.C., Latham, J.A., Vermaas, E.H. & Toole, J.J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355, 564–566 (1992).

    Article  CAS  Google Scholar 

  32. Rezaie, A.R. Heparin-binding exosite of factor Xa. Trends Cardiovasc. Med. 10, 333–338 (2000).

    Article  CAS  Google Scholar 

  33. Rydel, T.J. et al. The structure of a complex of recombinant hirudin and human α-thrombin. Science 249, 277–280 (1990).

    Article  CAS  Google Scholar 

  34. Niehrs, C., Huttner, W.B., Carvallo, D. & Degryse, E. Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases. J. Biol. Chem. 265, 9314–9318 (1990).

    CAS  PubMed  Google Scholar 

  35. Famulok, M., Blind, M. & Mayer, G. Intramers as promising new tools in functional proteomics. Chem. Biol. 8, 931–939 (2001).

    Article  CAS  Google Scholar 

  36. Cox, J.C. & Ellington, A.D. Automated selection of anti-protein aptamers. Bioorg. Med. Chem. 9, 2525–2531 (2001).

    Article  CAS  Google Scholar 

  37. Herschlag, D., Khosla, M., Tsuchihashi, Z. & Karpel, R.L. An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis. EMBO J. 13, 2913–2924 (1994).

    Article  CAS  Google Scholar 

  38. Robertson, M.P. & Ellington, A.D. In vitro selection of nucleoprotein enzymes. Nat. Biotechnol. 19, 650–655 (2001).

    Article  CAS  Google Scholar 

  39. Atsumi, S., Ikawa, Y., Shiraishi, H. & Inoue, T. Design and development of a catalytic ribonucleoprotein. EMBO J. 20, 5453–5460 (2001).

    Article  CAS  Google Scholar 

  40. Nissen, P., Hansen, J., Ban, N., Moore, P.B. & Steitz, T.A. The structural basis of ribosome activity in peptide bond synthesis. Science 289, 920–930 (2000).

    Article  CAS  Google Scholar 

  41. Valadkhan, S. & Manley, J.L. Splicing-related catalysis by protein-free snRNAs. Nature 413, 701–707 (2001).

    Article  CAS  Google Scholar 

  42. Herschlag, D. RNA chaperones and the RNA folding problem. J. Biol. Chem. 270, 20871–20874 (1995).

    Article  CAS  Google Scholar 

  43. Robertson, M.P. & Ellington, A.D. Design and optimization of effector-activated ribozyme ligases. Nucleic Acids Res. 28, 1751–1759 (2000).

    Article  CAS  Google Scholar 

  44. Jose, A.M., Soukup, G.A. & Breaker, R.R. Cooperative binding of effectors by an allosteric ribozyme. Nucleic Acids Res. 29, 1631–1637 (2001).

    Article  CAS  Google Scholar 

  45. Vitiello, D., Pecchia, D.B. & Burke, J.M. Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer. RNA 6, 628–637 (2000).

    Article  CAS  Google Scholar 

  46. Geiger, C. et al. Cytohesin-1 regulates β-2 integrin-mediated adhesion through both ARF-GEF function and interaction with LFA-1. EMBO J. 19, 2525–2536 (2000).

    Article  CAS  Google Scholar 

  47. Mayer, G. et al. Controlling small guanine-nucleotide-exchange factor function through cytoplasmic RNA intramers. Proc. Natl. Acad. Sci. USA 98, 4961–4965 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (to M.F.) and by National Institutes of Health grants AI-36083 and GM-61789 (to A.D.E.). We thank Julian Davies (University of Vancouver) for providing the antibiotics library, Christoph Müller (European Molecular Biology Laboratory, Grenoble) for NFκB, p52, and Bcl-3, Tobias Restle (Max-Planck-Insititut für Molekulare Physiologie, Dortmund) for HIV-RT, Günter Mayer for helpful discussions, and Michael Hoch and Waldemar Kolanus (University of Bonn) for comments on the manuscript. The following reagents were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases): HIV-1 Tat protein from John Brady and H9 cells and HIV-1IIIb from Robert Gallo.

Author information

Authors and Affiliations

  1. Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, Bonn, 53121, Germany

    Jörg S. Hartig, S. Hani Najafi-Shoushtari, Imke Grüne & Michael Famulok

  2. Department of Chemistry and Biochemistry University of Texas, University of Texas, ICMB A4800/MBB 3.448BA, 26th and Speedway, Austin, 78712, TX

    Amy Yan & Andrew D. Ellington

Authors
  1. Jörg S. Hartig
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Hani Najafi-Shoushtari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Imke Grüne
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amy Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew D. Ellington
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Famulok
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Famulok.

Ethics declarations

Competing interests

A patent application on this work has been submitted on behalf of Nascacell GmbH, (Tutzing, Germany).

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hartig, J., Najafi-Shoushtari, S., Grüne, I. et al. Protein-dependent ribozymes report molecular interactions in real time. Nat Biotechnol 20, 717–722 (2002). https://doi.org/10.1038/nbt0702-717

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0702-717

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing