Specific inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: Penetration and localization of oligonucleotides in clone 76 cells
Specific inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: Penetration and localization of oligonucleotides in clone 76 cells
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DOI：
10.1006/bbrc.1997.6185
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发表时间：
1997-03-17
影响因子：
3.1
通讯作者：
Takaku, H
中科院分区：
文献类型：
--
作者：
Hatta, T;Takai, K;Takaku, H
合成了具有四个靶位点（PB 1、PB 2、PA和NP）的脂质体包封的硫代磷酸酯寡核苷酸，并使用克隆76细胞系通过CAT-ELISA测定测试了抑制作用。与PB 2-AUG和PA-AUG起始密码子互补的硫代磷酸寡核苷酸（S-ODNs）具有高度的抑制作用。就流感病毒RNA聚合酶和NP的抑制而言，研究了靶AUG起始密码子序列向反义硫代磷酸寡核苷酸上的3 '端、5'端和/或中心位点的置换。在寡核苷酸的中心位点含有AUG起始密码子的反义硫代寡核苷酸具有最高的抑制效果。脂质体包封的硫代磷酸酯寡核苷酸表现出比游离寡核苷酸更高的抑制活性。通过共聚焦激光扫描显微镜观察用内包封的反义磷酸二酯寡核苷酸FITC-ODNs-PB 2-T3处理的克隆76细胞，显示明显在细胞质内的弥漫荧光。有趣的是，内包封的反义硫代磷酸寡核苷酸，FITC-S-ODNs-PB 2-T3在克隆76细胞的核区域中积累。然而，在用游离的反义硫代磷酸寡核苷酸处理的克隆76细胞的内体和胞浆中观察到弱荧光。(C)北京：科学出版社.
Liposomally encapsulated phosphorothioate oligonucleotides with four target sites (PB1, PB2, PA, and NP) were synthesized and tested for inhibitory effects by a CAT-ELISA assay using the clone 76 cell line. The liposomally encapsulated phosphorothioate oligonucleotides (S-ODNs) complementary to the sites of the PB2-AUG and PA-AUG initiation codons showed highly inhibitory effects. Displacement of the target AUG initiation codon sequence to the 3'-end, 5'-end, and/or center sites on the antisense phosphorothioate oligonucleotides was studied with regard to the inhibition of influenza virus RNA polymerases and NP. The antisense phosphorothioate oligonucleotide containing the AUG initiation codon at the center site of the oligonucleotide had the highest inhibitory effects. The liposomally encapsulated phosphorothioate oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. Observation of clone 76 cells treated with the endocapsulated antisense phosphodiester oligonucleotide, FITC-ODNs-PB2-T3, by a confocal laser scanning microscope, revealed diffuse fluorescence, apparently within the cytoplasm. Interestingly, the endocapsulated antisense phosphorothioate oligonucleotide, FITC-S-ODNs-PB2-T3 accumulated in the nuclear region of clone 76 cells. However, weak fluorescence was observed in the endosomes and in the cytoplasms of the clone 76 cells treated with the free antisense phosphorothioate oligonucleotides. (C) 1997 Academic Press.