Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis
| Autor: | Lina Prasmickaite, Øystein Fodstad, John Goodchild, Kristian Berg, Anders Høgset, Gunhild Mari Mælandsmo, Thomas A. Perkins, Eivind Hovig |
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| Rok vydání: | 1998 |
| Předmět: |
RNA Caps
media_common.quotation_subject Transfection Methylation Fatty Acids Monounsaturated chemistry.chemical_compound Tumor Cells Cultured Genetics Fluorescence microscope Humans RNA Catalytic Internalization Melanoma Fluorescent Dyes media_common Oligoribonucleotides Base Sequence biology Vesicle Ribozyme Electrophoresis Capillary Biological Transport Quaternary Ammonium Compounds Kinetics Cytosol Biochemistry chemistry biology.protein Thymidine Intracellular Research Article |
| Zdroj: | Nucleic Acids Research. 26:4241-4248 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/26.18.4241 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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