Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis.
The uptake and cellular metabolism of a fluorescein-labelled synthetic ribozyme stabilized by 2'- O -methyl modification and a 3' inverted thymidine have been studied, employing capillary gel electrophoresis as a novel and efficient analytical method. After internalization by DOTAP transfection, electrophoretic peaks of intact ribozyme and different degradation products were easily resolved and the amount of intracellular intact ribozyme was quantified to >10(7) molecules/cell at the peak value after 4 h transfection. On further incubation the amount of intracellular intact ribozyme decreased due to both degradation and efflux from the cell. However, even after 48 h incubation there were still >10(6) intact ribozyme molecules/cell. Clear differences both in uptake and in metabolism were seen when comparing DOTAP transfection with the uptake of free ribozyme. Fluorescence microscopy studies indicated that the ribozyme was mainly localized in intracellular granules, probably not accessible to target mRNA. This implies that agents able to release the intact ribozyme from intracellular vesicles into the cytosol should have a considerable potential for increasing the biological effects of synthetic ribozymes.
Department of Biophysics and Department of Tumour Biology, Institute for Cancer Research,The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. lina.prasmickaite@labmed.uio.no
L
L
Prasmickaite
A
A
Hogset
G
G
Maelandsmo
K
K
Berg
J
J
Goodchild
T
T
Perkins
O
O
Fodstad
E
E
Hovig
D001483:Base Sequence; D001692:Biological Transport; D019075:Electrophoresis, Capillary / Q000379:methods; D005229:Fatty Acids, Monounsaturated / Q000493:pharmacokinetics*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008545:Melanoma; D008745:Methylation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics*; D012315:RNA Caps / Q000737:chemistry; D016337:RNA, Catalytic / Q000378:metabolism* / Q000493:pharmacokinetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D009843:Oligoribonucleotides; D000644:Quaternary Ammonium Compounds; D012315:RNA Caps; D016337:RNA, Catalytic; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane