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:

In vitro selection of an allosteric ribozyme that transduces analytes to amplicons

Nature Biotechnology volume 17, pages 62–66 (1999)Cite this article

Abstract

We have selected an allosteric ribozyme ligase from a random sequence population that is activated up to 10,000-fold by oligonucleotide effectors. The ribozyme conforms to a classic two-state model for allostery in which the equilibrium between inactive and active conformers is dramatically altered by the presence of effector ligands. In the presence of the effector the allosteric ribozyme ligase generates templates that can subsequently be amplified using conventional amplification technologies, such as RT-PCR. Thus, the allosteric ribozyme can transduce (or convert) analytes into amplicons. We demonstrate two potential diagnostic applications of the selected allosteric ribozyme ligase: 'counting' short oligonucleotide effectors by RT-PCR, and counting a non-nucleic acid effector, ATP, by ligation.

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: N90 design and selection scheme.
Figure 2: Proposed secondary structural models for active (A) and inactive (B) conformations of the L1 ribozyme.
Figure 3: Nuclease probing of the L1 ribozyme.
Figure 4: Detection of an oligonucleotide effector.
Figure 5: Detection of a nonoligonucleotide effector.

Similar content being viewed by others

References

  1. Porta, H. and Lizardi, P.M. 1995. An allosteric hammerhead ribozyme. Bio/Technology 13: 161–164.

    Google Scholar 

  2. Tang, J. and Breaker, R.R. 1997. Rational design of allosteric ribozymes. Chem. Biol. 4: 453–459.

    Article  Google Scholar 

  3. Tang, J. and Breaker, R.R. 1997. Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection. RNA 3: 914–925.

    Google Scholar 

  4. Tang, J. and Breaker, R.R. 1998. Mechanism for allosteric inhibition of an ATP–sensitive ribozyme. Nucleic Acids Res. 26: 4214–4221.

    Article  Google Scholar 

  5. Araki, M., Okuno, Y., Hara, Y., and Sugiura, Y. 1998 . Allosteric regulation of a ribozyme activity through ligand–induced conformational change. Nucleic Acids Res. 26: 3379–3384.

    Article  Google Scholar 

  6. Jaeger, L. 1997. The new world of ribozymes. Curr. Opin. Struct. Biol. 7: 324–335.

    Article  Google Scholar 

  7. Breaker, R.R. 1997. In vitro selection of catalytic polynucleotides. Chem. Rev. 97: 371–390.

    Article  Google Scholar 

  8. Pan, T. 1997. Novel and variant ribozymes obtained through in vitro selection. Curr. Opin. Chem. Biol. 1: 17– 25 .

    Article  Google Scholar 

  9. Tanner, M.A., Anderson, E.M., Gutell, R.R., and Cech, T.R. 1997. Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes. RNA 3: 1037–1051 .

    Google Scholar 

  10. Wang, J.F., Downs, W.D., and Cech, T.R. 1993. Movement of the guide sequence during RNA catalysis by a group I ribozyme. Science 260: 504–508.

    Article  Google Scholar 

  11. Herschlag, D. and Khosla, M. 1994. Comparison of pH dependencies of the Tetrahymena ribozyme reactions with RNA 2´–substituted and phosphorothioate substrates reveals a rate–limiting conformational step. Biochemistry 33: 5291– 5297.

    Article  Google Scholar 

  12. Costa, M., Deme, E., Jacquier, A., and Michel, F. 1997. Multiple tertiary interactions involving domain II of group II self–splicing introns. J. Mol. Biol. 267: 520– 536.

    Article  Google Scholar 

  13. Bartel, D.P. and Szostak, J.W. 1993. Isolation of new ribozymes from a large pool of random sequences. Science 261: 1411–1418 .

    Article  Google Scholar 

  14. Hager, A.J. and Szostak, J.W. 1997. Isolation of novel ribozymes that ligate AMP–activated RNA substrates. Chem. Biol. 4: 607–617.

    Article  Google Scholar 

  15. Cuenoud, B. and Szostak, J.W. 1995. A DNA metalloenzyme with DNA ligase activity. Nature 375: 611–614.

    Article  Google Scholar 

  16. Ekland, E.H., Szostak, J.W., and Bartel, D.P. 1995. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269: 364–370 .

    Article  Google Scholar 

  17. Creighton, T.E. 1993. Proteins: structures and molecular properties , W.H. Freeman and Co., New York.

    Google Scholar 

  18. Scott, J.D., Fischer, E.H., Demaille, J.G., and Krebs, E.G. 1985. Identification of an inhibitory region of the heat–stable protein inhibitor of the cAMP–dependent protein kinase. Proc. Natl. Acad. Sci. USA 82: 4379– 4383.

    Article  Google Scholar 

  19. House, C. and Kemp, B.E. 1987. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science 238: 1726–1728.

    Article  Google Scholar 

  20. Poteet–Smith, C.E., Shabb, J.B., Francis, S.H., and Corbin, J.D. 1997. Identification of critical determinants for autoinhibition in the pseudosubstrate region of type Iα cAMP–dependent protein kinase. J. Biol. Chem. 272: 379– 388.

    Article  Google Scholar 

  21. Guatelli, J.C., Whitfield, K.M., Kwoh, D.Y., Barringer, K.J., Richman, D.D., and Gingeras, T.R. 1990. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87: 1874– 1878.

    Article  Google Scholar 

  22. Sassanfar, M. and Szostak, J.W. 1993. An RNA motif that binds ATP. Nature 364: 550 –553.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from Intelligene.

Author information

Authors and Affiliations

  1. Department of Chemistry and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, 78712, TX

    Michael P. Robertson & Andrew Ellington

Authors
  1. Michael P. Robertson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew Ellington
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew Ellington.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Robertson, M., Ellington, A. In vitro selection of an allosteric ribozyme that transduces analytes to amplicons. Nat Biotechnol 17, 62–66 (1999). https://doi.org/10.1038/5236

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/5236

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