Optimization of Hammerhead Ribozymes for the Cleavage of S100A4 (CAPL) mRNA

Abstract

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6 + 8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6 + 6) and (5 + 5) nt. Stimulation was particularly strong in the case of the (5 + 5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.