Nucleic Acids Research, Vol 27, Issue 15 3159-3167, Copyright © 1999 by Oxford University Press
B Bramlage, S Alefelder, P Marschall and F Eckstein
Two synthetic hammerhead ribozymes, one unmodified and the other with
2"-modifications and four phosphorothioate groups, targeting a single GUA
site in the luciferase mRNA, were compared for their inhibition of gene
expression in cell cultureand their cellular uptake was also analysed. A
HeLa X1/5 cell line stably expressing luciferase, under an inducible
promoter, was treated with these ribozymes by liposome- mediated
transfection to determine their activity.Luciferase expression in cells was
inhibited to approximately 50% with little difference between the
unmodified and the 2"-modified ribozyme. A similar degree of inhibition was
observed with two catalytically inactive ribozymes, indicating that
inhibition was mainly due to an antisense effect. A ribozyme carrying a
cholesterol moiety, applied to the cells without carrier, showed no
inhibition. Northern blotting indicated a similar amount of cellular uptake
of all ribozymes. The unmodified ribozyme was essentially evenly
distributed between cytoplasm and nucleus, whereas a higher proportion of
the phosphorothioate-containing ribozyme was observed in the nucleus.
Fluorescence microscopy, including confocal microscopy using
5"-fluorescein-labelled ribozymes, showed that the unmodified and
2"-modified ribozymes 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 expression to a comparable degree, suggesting that the ribozyme
in the nucleus did not contribute significantly to the inhibition.
Ribozymes with a cholesterol moiety were predominantly trapped in the cell
membrane, explaining their inability to interfere with gene expression.
ARTICLES
Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake
Max-Planck-Institut fur experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Gottingen, Germany.
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