Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake

Two synthetic hammerhead ribozymes, one unmodified and the other with 2'-mo difications and four phosphorothioate groups, targeting a single GUA site i n the luciferase mRNA, were compared for their inhibition of gene expressio n in cell culture and their cellular uptake was also analysed. A HeLa X1/5 cell line stably expressing luciferase, under an inducible promoter, was tr eated with these ribozymes by liposome-mediated transfection to determine t heir activity. Luciferase expression in cells was inhibited to similar to 5 0% with little difference between the unmodified and the 2'-modified ribozy me, A similar degree of inhibition was observed with two catalytically inac tive ribozymes, indicating that inhibition was mainly due to an anti-sense effect, A ribozyme carrying a cholesterol moiety, applied to the cells with out carrier, showed no inhibition, Northern blotting indicated a similar am ount of cellular uptake of all ribozymes, The unmodified ribozyme was essen tially evenly distributed between cytoplasm and nucleus, whereas a higher p roportion of the phosphorothioate-containing ribozyme was observed in the n ucleus. Fluorescence microscopy, including confocal microscopy using 5'-flu orescein-labelled ribozymes, showed that the unmodified and 2'-modified rib ozymes were present in the cytoplasm and in the nucleus to a similar extent , whereas the fluorescence of the phosphorothioate-containing ribozyme was much stronger in the nucleus. Both ribozymes inhibited luciferase expressio n to a comparable degree, suggesting that the ribozyme in the nucleus did n ot contribute significantly to the inhibition. Ribozymes with a cholesterol moiety were predominantly trapped in the cell membrane, explaining their i nability to interfere with gene expression.