A COMPARISON OF THE IN-VITRO ACTIVITY OF DNA-ARMED AND ALL-RNA HAMMERHEAD RIBOZYMES

Hammerhead ribozymes targeted against two unrelated RNA substrates hav e been prepared. For each substrate, four ribozymes, differing in thei r hybridising arm length and composition (DNA or RNA), have been synth esised and kinetically characterised. The presence of DNA in the hybri dising arms had little effect on the overall cleavage rate when the cl eavage step was rate determining. Shortening each of the hybridising a rms of ribozymes from 10 to 6 nucleotides generally resulted in modest changes in rate constants for cleavage of the same 13mer substrate. I n one case the presence of long RNA hybridising arms significantly imp eded the cleavage reaction, Cleavage rates displayed first order depen dence on hydroxide ion concentration at low pHs, At higher pH, some ri bozymes deviated from this first order dependence because of a change in the rate-determining step, possibly due to a requirement for a conf ormation change in the ribozyme-substrate complex prior to cleavage. R ibozyme cleavage was strongly dependent on temperature in the range 5- 45 degrees C, with an activation energy for the reaction of similar to 60 kJ mol(-1). The ribozymes displayed biphasic dependence on magnesi um ion concentration; evidence of strong apparent binding (K-d similar to 10 mM) as well as a looser interaction was observed for all ribozy mes.