Previously, suppression of the S100A4 mRNA by an endogenously expressed rib
ozyme in osteosarcoma cells was shown to inhibit their metastasis in rats.
As a prelude to performing similar studies with exogenous, synthetic ribozy
mes, we compared a series of hammerhead ribozymes targeted against differen
t sites in the mRNA, The ribozymes differed only in the 7-base flanking seq
uences complementary to the substrate and were protected against nucleases
by chemical modification. Cleavage efficiency varied widely and was not obv
iously related to the predicted secondary structure of the target RNA. The
most active ribozyme of the series was chosen for further optimization. Len
gthening 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 nucleotide
s (nt) in stem III and 8 nt in stem I. Although these stems strongly influe
nce ribozyme performance, their optimization is still empirical. Faster cle
avage was obtained by adding facilitator oligonucleotides to ribozymes with
shorter stems of (6+6) and (5+5) nt, Stimulation was particularly strong i
n the case of the (5+5) ribozyme, which was poorly active by itself. The en
hancement caused by different facilitator oligonucleotides paralleled their
expected ability to hybridize to RNA as a function of length and chemical
modification.