Marcusson E G, Bhat B, Manoharan M, Bennett C F, Dean N M
Isis Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA.
Nucleic Acids Res. 1998 Apr 15;26(8):2016-23. doi: 10.1093/nar/26.8.2016.
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.
与特定mRNA序列互补的反义寡核苷酸是体外和体内广泛使用的基因表达抑制剂。在体外，阳离子脂质已被证明可通过增加细胞摄取和促进核内积累来提高反义寡核苷酸的药理活性。我们使用荧光标记的脂质和靶向蛋白激酶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在细胞表面和细胞质内的微小点状结构中共定位。在随后的时间里，寡核苷酸开始在细胞核以及细胞质中积累。相比之下，阳离子脂质仍定位于细胞表面和细胞质，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。这也与一种较小的mRNA转录本的短暂出现同时发生，据信这是由蛋白激酶C-α mRNA的RNase H切割导致的。这些数据表明，尽管阳离子脂质促进了寡核苷酸的摄取，但在寡核苷酸能够进入细胞核并诱导靶向mRNA降解之前，复合物必须解离。
