Kuwasaki T, Hosono K, Takai K, Ushijima K, Nakashima H, Saito T, Yamamoto N, Takaku H
Department of Industrial Chemistry, Chiba Institute of Technology, Narashino, Japan.
Biochem Biophys Res Commun. 1996 Nov 12;228(2):623-31. doi: 10.1006/bbrc.1996.1707.
Hairpin antisense oligodeoxyribonucleotides containing 2'-methoxynucleosides were more active in the micromolar concentration range than linear and DNA hairpin phosphorothioate oligonucleotides with the same sequence. Furthermore, the abilities of hairpin antisense and random hairpin phosphorothioate oligonucleotides to inhibit HIV-1 replication were examined. Antisense oligonucleotides inhibit the replication and the expression of HIV-1 more efficiently than random-oligomers of the same length or with the same internucleotide modification. Four different target sites (gag, pol, rev, and tat) within the HIV-1 genome were studied with regard to the inhibition of HIV-1 replication by antisense oligonucleotides. Antisense oligomers complementary to the sites of the initiation sequences of gag were most effective. The [32P]-labeled hairpin phosphorothioate oligonucleotide was rapidly assimilated by MOLT-4 cells, whereas the [32P]-labeled hairpin phosphodiester oligonucleotide was not. In MOLT-4 cells treated with the FITC-hairpin phosphorothioate oligonucleotide containing 2'-methoxynucleosides by a confocal laser scanning microscope, diffuse fluorescence was observed in the cytoplasm. Interestingly, fluorescent signals accumulated in the nuclear region of chronically infected MOLT-4/HIV-1 cells after a 60 min incubation.
含有2'-甲氧基核苷的发夹反义寡脱氧核糖核苷酸在微摩尔浓度范围内比具有相同序列的线性和DNA发夹硫代磷酸酯寡核苷酸更具活性。此外，还研究了发夹反义寡核苷酸和随机发夹硫代磷酸酯寡核苷酸抑制HIV-1复制的能力。反义寡核苷酸比相同长度或具有相同核苷酸间修饰的随机寡聚物更有效地抑制HIV-1的复制和表达。针对HIV-1基因组内的四个不同靶位点（gag、pol、rev和tat），研究了反义寡核苷酸对HIV-1复制的抑制作用。与gag起始序列位点互补的反义寡聚物最为有效。[32P]标记的发夹硫代磷酸酯寡核苷酸能被MOLT-4细胞迅速摄取，而[32P]标记的发夹磷酸二酯寡核苷酸则不能。在用含2'-甲氧基核苷的FITC-发夹硫代磷酸酯寡核苷酸处理的MOLT-4细胞中，通过共聚焦激光扫描显微镜观察到细胞质中有弥漫性荧光。有趣的是，孵育60分钟后，荧光信号在慢性感染的MOLT-4/HIV-1细胞的核区域积累。
