Nucleic Acids Research, 1993, Vol. 21, No. 16 3857-3865
© 1993
MOLECULAR BIOLOGY |
Intracellular disposition and metabolism of fluorescently-labled unmodified and modified oligouncleotides microijjected into mammalian cells
Gilead Sciences 346 Lakeside Drive, Foster City, CA 94404, USA
*To whom correspondence should be addressed
Received March 10, 1993. Revised June 9, 1993. Accepted June 9, 1993.
The intracellular distribution and metabolism of micro- Injected fluorescently-labeled oligonucleotides (ODNs) have been evaluated using confocal fluorescence microscopy. Fluorescent phosphodiester ODNs, micro- Injected Into the cytoplasm of mammalian cells, rapidly accumulate within the nucleus; the fluorescence disappears with a half-life of 15 —20 minutes. Micro- Injected fluorescent phosphorothioate ODNs remain In the nucleus for more than 24 hours. The persistence of fluorescence depends on the length of the ODN. Modification of the 3' end of phosphodiester ODNs does not significantly slow the rapid disappearance of fluorescence, although certain 3' modifications localize ODNs into the cytoplasm. Using specially designed ODNs, endonuclease activity Is demonstrated to exist in the cytoplasm and nucleus. Modification of the 2' position of the ribose rings of a fluorescent phosphodiester ollgodeoxynucleotide with O-methyl or O-allyl does not alter its Intracellular distribution; however, the 2'-O-allyl modification stabilizes the persistence of fluorescence more than 60-fold compared to the 2'-deoxy control. Thus, the experiments Indicate that somatic cells contain nucleolytlc activities which degrade microinjected ODNs; however, chemical modification can dramatically circumvent this process.
+Present address: Program of Cellular and Developmental Biology, Harvard Medical School, Boston, MA 02115, USA
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