Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus
Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus
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DOI：
10.1093/nar/26.8.2016
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发表时间：
1998-04-15
影响因子：
14.9
通讯作者：
Dean, NM
中科院分区：
文献类型：
--
作者：
Marcusson, EG;Bhat, B;Dean, NM
与特定 mRNA 序列互补的反义寡核苷酸是广泛使用的体外和体内基因表达抑制剂。体外阳离子脂质已被证明可以通过增加细胞摄取和促进核积累来增加反义寡核苷酸的药理活性。我们使用荧光标记的脂质和针对蛋白激酶 C-α 的寡核苷酸研究了寡核苷酸/阳离子脂质复合物的细胞内命运。添加到细胞后，脂质最初与寡核苷酸共定位在细胞表面上并在细胞质内的精细点状结构中。后来，寡核苷酸开始在细胞核和细胞质中积累。相反，阳离子脂质仍然定位于细胞表面和细胞质，并且从未在细胞核中发现。直到在细胞核中看到寡核苷酸后，蛋白激酶 C-α mRNA 的表达才开始下降。这也与较小 mRNA 转录物的短暂出现相一致，该转录物被认为是由 RNase H 切割蛋白激酶 C-α mRNA 造成的。这些数据表明，虽然阳离子脂质促进寡核苷酸的摄取，但在寡核苷酸能够进入细胞核并诱导目标mRNA降解之前，复合物必须解离。
Antisense oligonucleotides complementary to specific mRNA sequences are widely used inhibitors of gene expression in vitro and in vivo. In vitro cationic lipids have been demonstrated to increase the pharmacological activity of antisense oligonucleotides by increasing cellular uptake and facilitating nuclear accumulation. We have investigated the intracellular fate of oligonucleotide/cationic lipid complexes using fluorescently labeled lipids and oligonucleotides targeted to protein kinase C-alpha. After addition to cells the lipids initially co-localized with the oligonucleotide on the cell surface and in fine punctate structures within the cytoplasm. At later times the oligonucleotide began to accumulate in the nucleus as well as the cytoplasm. In contrast, the cationic lipid remained localized to the cell surface and the cytoplasm and was never found in the nucleus. Expression of protein kinase C-alpha mRNA did not begin to decline until after oligonucleotide was seen in the nucleus. This was also coincident with the transient appearance of a smaller mRNA transcript believed to result from RNase H cleavage of protein kinase C-alpha mRNA. These data suggest that although cationic lipids facilitate uptake of oligonucleotides, the complex must disassociate before the oligonucleotide can gain access to the nucleus and induce degradation of targeted mRNA.