Inhibition of luciferase expression by synthetic hammerhead ribozymes and their cellular uptake.
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.
Bramlage
B
B
Max-Planck-Institut für experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Göttingen, Germany.
Alefelder
S
S
Marschall
P
P
Eckstein
F
F
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism; D015536:Down-Regulation; D004337:Drug Carriers; D019793:Fluorescein; D017930:Genes, Reporter / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D004337:Drug Carriers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D002784:Cholesterol; D008156:Luciferases; D019793:Fluorescein