TY - JOUR
T1 - Mechansism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages
AU - Shoji, Yoko
AU - Akhtar, Saghir
AU - Periasamy, Ammasi
AU - Herman, Brian
AU - Juliano, R. L.
N1 - Funding Information:
This work was supported by NIH grant
PY - 1991/10/25
Y1 - 1991/10/25
N2 - The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and luorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occuring between 15 and 20°C. Most of the label which becomes cell associated at 37°C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an ndosomal/lysosomal arker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonuclotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be locked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.
AB - The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and luorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occuring between 15 and 20°C. Most of the label which becomes cell associated at 37°C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an ndosomal/lysosomal arker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonuclotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be locked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.
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U2 - 10.1093/nar/19.20.5543
DO - 10.1093/nar/19.20.5543
M3 - Article
C2 - 1658734
AN - SCOPUS:0026040132
SN - 0305-1048
VL - 19
SP - 5543
EP - 5550
JO - Nucleic acids research
JF - Nucleic acids research
IS - 20
ER -