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.

  • Research Article
  • Published:

Creation of a ribonuclease abzyme through site-directed mutagenesis

Nature Biotechnology volume 16, pages 1065–1067 (1998)Cite this article

Abstract

The development of abzymes (antibody/enzymes) is one method of creating reagents with novel catalytic activity. To date, most abzymes have been obtained by immunization with transition state analogs. We have chosen to start with an existing antibody and convert it into an enzyme by the addition of catalytic residues to the binding site. We have introduced a histidine residue into antibody Jel 103 and converted it into an abzyme that cleaves poly(rI) with a kinetic efficiency of about 100 M–1sec–1.

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: Proposed mechanism of ribonuclease action for the His96H mutant, based on the mechanism of naturally occurring ribonucleases12,13.
Figure 2: Fluorescence polarization measurements of the binding of Jel 103 (▪), scFv.103 (●), and the His98H mutant scFv (♦) to fluorescein-labeled (rI)20, and scFv.103 (▴) to fluorescein-labeled (rC)20.
Figure 3: (A) Digestion of fluorescein-labeled (rI)20 assessed by fluorescence polarization. scFv.103 (▪), mutant His98H (▴), His96H (●).
Figure 4: Gel electrophoretic analysis of fluorescein-labeled (rI)20 incubated with His96H.
Figure 5: Digestion of poly(rI) by His96H (▴).

Similar content being viewed by others

References

  1. Tramontano, A., Janda, K.D., and Lerner, R.A. 1986. Catalytic antibodies. Science 234: 1566–1570.

    Article  Google Scholar 

  2. Pollack, S.J., Jacobs, J.W., and Schultz, P.G. 1986. Selective chemical catalysis by an antibody. Science 234: 1570– 1573.

    Article  Google Scholar 

  3. Raso, V. and Stollar, B.D. 1975. The antibody enzyme analogy: comparison of enzymes and antibodies specific for phosphopyridoxyltyrosine. Biochemistry 14: 591–599.

    Article  Google Scholar 

  4. Schultz, P.G. and Lerner, R.A. 1995. From molecular diversity to catalysis: lessons from the immune system. Science 269: 1835–1842.

    Article  Google Scholar 

  5. Kirby, A.J. 1996. Enzyme mechanisms, models, and mimics. Angew. Chem. Int. Ed. Engl. 35: 707– 724.

    Article  Google Scholar 

  6. Kirby, A.J. 1996. The potential of catalytic antibodies. Acta. Chem. Scand. 50: 203–210.

    Article  Google Scholar 

  7. Shuster, A.M., Golobov, G.V., Kvashuk, O.A., Bogomolova, A.E., Smirnov, I.V., and Gabibov, A.G. 1992. DNA-hydrolyzing autoantibodies. Science 256: 665–667.

    Article  Google Scholar 

  8. Paul, S., Volle, D.J., Beach, C.M., Johnson, D.R., Powell, M.J., and Massey, R.J. 1989. Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody. Science 244: 1158–1162.

    Article  Google Scholar 

  9. Stewart, J.D. and Benkovic, S.J. 1995. Transition-state stabilization as a measure of the efficiency of antibody catalysis. Nature 375: 388–391.

    Article  Google Scholar 

  10. Pokkuluri, P.R., Bouthillier, F., Li, Y., Kuderova, A., Lee, J., and Cygler, M. 1994. Preparation, characterization and crystallization of an antibody fab fragment that recognizes RNA. J. Mol. Biol. 243: 283–297.

    Article  Google Scholar 

  11. Kuderova, A., Tanha, J., and Lee, J.S. 1994. Characterization of four nucleic acid-binding single-chain Fv fragments by direct and competitive solid-phase radioimmunoassays. J. Biol. Chem. 269: 32957– 32962.

    Google Scholar 

  12. Breslow, R., Huang, D.L., and Anslyn, E. 1989. On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+. Proc. Natl. Acad. Sci. USA 86: 1746–1750.

    Article  Google Scholar 

  13. Gohda, K., Oka, K.-I., Tomita, K.-I., and Hakoshima, T. 1994. Crystal structure of RNase T1 complexed with the product nucleotide 3'-GMP. J. Biol. Chem. 269: 17531–17536.

    Google Scholar 

  14. Baldwin, E. and Schultz, P.G. 1989. Generation of a catalytic antibody by site-directed mutagenesis. Science 245: 1104–1107.

    Article  Google Scholar 

  15. Heyduk, T. and Lee, J.C. 1990. Application of fluorescence energy transfer and polarization to monitor Escherichia coli cAMP receptor protein and lac promoter interaction. Proc. Natl. Acad. Sci. USA 87: 1744– 1748.

    Article  Google Scholar 

  16. LeTilly, V. and Royer, C.A. 1993. Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding. Biochemistry 32: 7753–7758.

    Article  Google Scholar 

  17. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M. et al. 1988. Single-chain antigen binding proteins. Science 242: 423– 426.

    Article  Google Scholar 

  18. Glockshuber, R., Malia, M., Pfitzinger, I., and Plückthun, A. 1990. A comparison of strategies to stabilize immunoglobulin Fv-Fragments. Biochemistry 29: 1362–1367.

    Article  Google Scholar 

  19. Barry, M.M. and Lee, J.S. 1993. Cloning and expression of an autoimmune DNA-binding single chain Fv. Only the heavy chain is required for binding. Mol. Immunol. 30: 833–840.

    Article  Google Scholar 

  20. Jia, Z., Quail, J.W., Waygood, E.B., and Delbaere, L.T.J. 1987. The 2.0-Å resolution structure of Escherichia coli histidine-containing phosphocarrier protein HPr. J. Biol. Chem. 268: 22490–22501.

    Google Scholar 

  21. Prasad, L., Vandonselaar, M., Lee, J.S., and Delbaere, L.T.J. 1988. Structure determination of a monoclonal fab fragment specific for histidine-containing protein of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli. J. Biol. Chem. 263: 2571–2574.

    Google Scholar 

  22. Herron, J.N., He, X-H., Ballard, D.W., Blier, P.R., Pace, P.E., Bothwell, A.L.M. et al. 1991. An autoantibody to single-stranded DNA: comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex. Proteins: Struct. Funct. Genet. 11: 159– 175.

    Article  Google Scholar 

  23. Morgan, A.R., Lee, J.S., Pulleyblank, D.E., Murray, N.L., and Evans, D.H. 1979. Ethidium fluorescence assays. Nucleic Acids Res. 7: 547–569.

    Article  Google Scholar 

  24. Walz, F.G., Osterman, H.L., and Libertin, C. 1979. Base-group specificity at the primary recognition site of ribonuclease T1 for minimal RNA substrates. Arch. Biochem. Biophys. 195: 95–102.

    Article  Google Scholar 

  25. Mossakowska, D.E., Nyberg, K., and Fersht, A.R. 1989. Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (Barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry 28: 3843– 3850.

    Article  Google Scholar 

  26. Quéméneur, E., Moutiez, M., Charbonnier, J-B., and Ménez, A. 1998. Engineering cyclophilin into a proline-specific endopeptidase. Nature 391: 301– 303.

    Article  Google Scholar 

  27. Braun, R.P., Woodsworth, M.L., and Lee, J.S. 1986. The immunogenic properties of poly(dI).poly(dC) and poly(rI).poly(dC). Analysis of monoclonal antibodies. Mol. Immunol. 23: 685– 691.

    Article  Google Scholar 

  28. Tanha, J. and Lee, J.S. 1997. Thermodynamic analysis of monoclonal antibody binding to duplex DNA. Nucl. Acids Res. 25: 1442–1449.

    Article  Google Scholar 

Download references

Acknowledgements

This work was funded by Medical Research Council by grants to J.S.L. and by Health Services Utilization and Research Commission for fellowships for M.C.F. and A.K.

Author information

Authors and Affiliations

  1. Laboratory of Plant Molecular Physiology, Masaryk University , 611 37 Brno, Kotlarska, 2, Czech Republic

    Alena Kuderova

  2. Biotechnology Research Institute, National Research Council of Canada, Montreal, H4P 2R2 , Quebec, Canada

    Miroslaw Cygler

Authors
  1. Michael C. Fletcher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alena Kuderova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miroslaw Cygler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeremy S. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fletcher, M., Kuderova, A., Cygler, M. et al. Creation of a ribonuclease abzyme through site-directed mutagenesis. Nat Biotechnol 16, 1065–1067 (1998). https://doi.org/10.1038/3519

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/3519

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