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Nucleic Acids Research, 1992, Vol. 20, No. 21 5737-5741
© 1992

MOLECULAR BIOLOGY

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability

Philip Hendry*, Maxine J. McCall, Fernando S. Santiago and Philip A. Jennings+

CSIRO, Division of Biomolecular Engineering PO Box 184 North Ryde, NSW 2113, Australia

* To whom correspondence should be addressed

Received July 6, 1992. Revised October 6, 1992. Accepted October 6, 1992.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising "arms’ of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150µM–1min–1 for the RNA-and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.

+ Present address: CSIRO. Division of Tropical Animal Production, Private Bag No. 3, PO Indooroopilly. Queensland 4068, Australia


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