IN-VITRO ACTIVITY OF MINIMIZED HAMMERHEAD RIBOZYMES

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