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Published online 15 June 2004

Nucleic Acids Research, 2004, Vol. 32, No. 10 3212-3219
© 2004 Nucleic Acids Research, Vol. 32 No. 10 © Oxford University Press 2004; all rights reserved

Sensing complex regulatory networks by conformationally controlled hairpin ribozymes

S. Hani Najafi-Shoushtari, Günter Mayer and Michael Famulok*

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

* To whom correspondence should be addressed. Tel: +49 228 73 1787; Fax: +49 228 73 5338; Email: m.famulok{at}uni-bonn.de

Received April 15, 2004; Revised and Accepted May 16, 2004

The hairpin ribozyme catalyses RNA cleavage by a mechanism utilizing its conformational flexibility during the docking of two independently folded internal loop domains A and B. Based on this mechanism, we designed hairpin ribozyme variants that can be induced or repressed by external effector oligonucleotides influencing the docking process. We incorporated a third domain C to assimilate alternate stable RNA motifs such as a pseudo-half-knot or an internal stem–loop structure. Small sequence changes in domain C allowed targeted switching of ribozyme activity: the same effector oligonucleotide can either serve as an inducer or repressor. The ribozymes were applied to trp leader mRNA, the RNA sequence tightly bound by L-tryptophan-activated trp-RNA-binding attenuation protein (TRAP). When domain C is complementary to this mRNA, ribozyme activity can be altered by annealing trp leader mRNA, then specifically reverted by its TRAP/tryptophan-mediated sequestration. This approach allows to precisely sense the activity status of a protein controlled by its metabolite molecule.


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