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Nucleic Acids Research, 2000, Vol. 28, No. 4 886-894
© 2000 Oxford University Press

RNA double cleavage by a hairpin-derived twin ribozyme

Christian Schmidt, Rüdiger Welz and Sabine Müller*

Humboldt-Universität zu Berlin, Institut für Chemie, Fachinstitut für Organische und Bioorganische Chemie, Hessische Straße 1-2, 10115 Berlin, Germany

The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.

* To whom correspondence should be addressed. Tel: +49 30 20938393; Fax: +49 30 20938479; Email: sabine=mueller@chemie.hu-berlin.de


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