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Nucleic Acids Research, 1995, Vol. 23, No. 19 3922-3927
© 1995

RNA

In vitro activity of minimised hammerhead ribozymes

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

CSIR0, Division of Biomolecular Engineering PO Box 184, North Ryde, NSW 2113, Australia

*To whom correspondence should be addressed

Received June 21, 1995. Revised August 30, 1995. Accepted August 30, 1995.

A number of minimised hammerhead ribozymes (minizymes) which lack stem II have been kinetically characterised. These minizymes display optimal cleavage activity at temperatures around 37°C. The cleavage reactions of the minizymes are first order in hydroxide ion concentration up to around pH 9.3 above which the cleavage rate constants decline rapidly. The reactions show a biphasic dependence on magnesium-ion concentration; one of the interactions has an apparent dissociation constant of around 20 mM while the other appears to be very weak, showing no sign of saturation at 200 mM MgCl2 The minizymes are significantly less active than comparable, full-size ribozymes when cleaving short substrates. However, at a particular site in a transcribed TAT gene from HIV-1, minizymes are more effective than ribozymes.

Present addresses: R. W. Johnson Pharmaceutical Research Institute, GPO Box 3331, Sydney, NSW 2001, Australia and

CSLRO, Division of Tropical Animal Production, Private Bag No.3, Indooroopilly, QLD 44)68, Australia.


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