ENHANCEMENT OF THE CLEAVAGE RATES OF DNA-ARMED HAMMERHEAD RIBOZYMES BY VARIOUS DIVALENT METAL-IONS

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all R NA ribozyme. Determined k(cat) values were proven to represent the che mical-cleavage step and not the product-dissociation step. In agreemen t with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464 - 9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic r adius of 0.65 and 1.0 angstrom, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic paramete rs in the presence of various divalent metal ions revealed that DNA ar ms always enhanced k(cat) values. Chemical-probing data using dimethyl sulfate indicated that the catalytic-loop structures of all-RNA and ch imeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chi meric ribozyme. These observations and others demonstrate that DNA sub stitution in non-catalytic-loop regions increases chemical-cleavage ac tivity, possibly with an accompanying very subtle change in the struct ure.