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In Vitro Selection of Allosteric Ribozymes

  • Nicolas Piganeau
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 535)

Abstract

In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. The present protocol describes an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to virtually any drug of choice. We applied this method to select hammerhead ribozymes inhibited specifically by doxycycline or pefloxacin in the sub-micromolar range. The selected ribozymes can be converted into classical aptamers via insertion of a point mutation in the catalytic center of the ribozyme.

Keywords

Ribozyme in vitro selection allostery aptazyme 

References

  1. 1.
    Breaker, R.R. (2002) Engineered allosteric ribozymes as biosensor components. Curr. Opin. Biotechnol. 13, 31–39.PubMedCrossRefGoogle Scholar
  2. 2.
    Yen, L., Svendsen, J., Lee, J. S., Gray, J.T., Magnier, M., Baba, T., D'Amato, R.J. and Mulligan, R.C. (2004) Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431, 471–476.PubMedCrossRefGoogle Scholar
  3. 3.
    Tang, J. and Breaker, R.R. (1997) Rational design of allosteric ribozymes. Chem. Biol. 4, 453–459.PubMedCrossRefGoogle Scholar
  4. 4.
    Koizumi, M., Soukup, G.A., Kerr, J.N. and Breaker, R.R. (1999) Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat. Struct. Biol. 6, 1062–1071.PubMedCrossRefGoogle Scholar
  5. 5.
    Piganeau, N., Jenne, A., Thuillier, V. and Famulok, M. (2001) An allosteric ribozyme regulated by doxycyline. Angew. Chem. Int. Ed. Engl. 40, 3503.PubMedCrossRefGoogle Scholar
  6. 6.
    Piganeau, N., Thuillier, V. and Famulok, M. (2001) In vitro selection of allosteric ribozymes: theory and experimental validation. J. Mol. Biol. 312, 1177–1190.PubMedCrossRefGoogle Scholar
  7. 7.
    Cadwell, R.C. and Joyce, G.F. (1994) Mutagenic PCR. PCR Methods Appl. 3, S136–S140.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Nicolas Piganeau
    • 1
  1. 1.Department of Chemistry, Institute for Biochemistry and Molecular BiologyUniversity of HamburgHamburgGermany

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