Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.
Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.
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细胞摄取含有甲基膦酸键的修饰寡脱氧核苷酸的机制。
DOI：
10.1093/nar/19.20.5543
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发表时间：
1991
影响因子：
14.9
通讯作者：
Juliano,RL
中科院分区：
文献类型：
--
作者：
Shoji,Y;Akhtar,S;Periasamy,A;Herman,B;Juliano,RL
用~（32）P标记和荧光标记的寡核苷酸研究了甲基膦酸酯寡核苷酸（15聚体）的细胞摄取和细胞内分布。甲基膦酸酯寡核苷酸的细胞摄取过程高度依赖于温度，在15 ° C至20°C之间摄取显著增加。大多数在37°C下与细胞结合的标记不能通过酸洗或胰蛋白酶消化去除，因此似乎在细胞内。可视化的罗丹明标记的甲基膦酸酯寡核苷酸使用数字成像荧光显微镜揭示了一个泡状的亚细胞分布暗示的内体定位。罗丹明标记的甲基膦酸酯寡核苷酸与荧光素-葡聚糖（一种核内体/溶酶体标记物）存在广泛的共定位。标记的甲基膦酸酯寡核苷酸的表观内吞摄取不能通过与未标记的甲基膦酸酯或磷酸二酯寡核苷酸的竞争来阻断，也不能通过ATP来阻断。这与放射性标记的磷酸二酯寡核苷酸的情况相反，其摄取可以被未标记的竞争剂完全阻断。磷酸二酯寡核苷酸的摄取，但不是甲基膦酸酯寡核苷酸，可以锁定酸化的细胞质。这些观察结果表明，甲基膦酸酯寡核苷酸的细胞摄取途径涉及液相或吸附性内吞作用，并且不同于磷酸二酯寡核苷酸的摄取途径。
The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both32P 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.
影响因子：
3.7
作者：
H. Petty
通讯作者：
H. Petty