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Nucleic Acids Research, 1994, Vol. 22, No. 24 5271-5278
© 1994

Articles

The influence of imperfectly paired helices I and III on the catalytic activity of hammerhead ribozymes

Michalis Zoumadakis, Wolfgang J. Neubert1 and Martin Tabler*,

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas PO Box 1527, GR-71110 Heraklion, Crete, Greece 1Abteilung fur Virusforschung, Max-Planck-lnstitut fur Biochemie D-82152 Martinsried, Germany

*To whom correspondence should be addressed

Received September 7, 1994. Accepted November 2, 1994.

Several catalytic antisense RNAs directed against different regions of the genomic or antlgenomlc RNA of Sendai virus were constructed. All RNAs contained the same catalytic domain based on hammerhead ribozymes but some had deletions or mutations resulting In Imperfect helices I and III. Pre-annealed substrate/ribozyme complexes were used to determine the rates of the cleavage process for the different ribozymes under single-turnover conditions. It was found that the sequence context surrounding the cleavable motif influenced the cleavage efficiencies. Deletions or mutations of nucleotides 2.1 or 15.1 and 15.2 according to the numbering system for hammerhead ribozymes of Hertel et al. (1) destroyed catalytic activity. Deletions of nucleotide 2.2 or additional nucleotides In the helix l-formlng region oi the ribozyme did not destruct, but only reduced the cleavage efficiencies. Similar results were observed for a deletion of nucleotide 15.3. Simultaneous deletions within helices I and III resulted In alternative cleavage sites. The potential consequences for the specificity of the ribozyme reaction are discussed.


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