EFFECTS OF PHOSPHOROTHIOATE CAPPING ON ANTISENSE OLIGONUCLEOTIDE STABILITY, HYBRIDIZATION AND ANTIVIRAL EFFICACY VERSUS HERPES-SIMPLEX VIRUS-INFECTION
EFFECTS OF PHOSPHOROTHIOATE CAPPING ON ANTISENSE OLIGONUCLEOTIDE STABILITY, HYBRIDIZATION AND ANTIVIRAL EFFICACY VERSUS HERPES-SIMPLEX VIRUS-INFECTION
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DOI：
10.1093/nar/19.20.5743
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发表时间：
1991-10-25
影响因子：
14.9
通讯作者：
ECKER, DJ
中科院分区：
文献类型：
--
作者：
HOKE, GD;DRAPER, K;ECKER, DJ
已经努力通过向寡核苷酸的3'、5'或3'和5'末端添加各种修饰来改善磷酸二酯(PO)寡核苷酸的生物稳定性。 ISIS 1080 是一种硫代磷酸酯 (PS) 21 聚体寡核苷酸，与 HSV-1 中 UL13 mRNA 的内部 AUG 密码子互补，可将 HeLa 细胞中 HSV-1 的感染率降低至 9.0% +/- 11%。与完全 PS ISIS 1080 相比，在 3' (ISIS 1365)、5'(ISIS 1370)、3' 和 5'(ISIS 1364) 末端含有三个 PS 连接的 ISIS 1080 PO 类似物或在中间具有四个连接 (ISIS 1400) 的抗病毒功效降低。热变性曲线表明这些寡核苷酸与互补 DNA 杂交或 具有同等结合亲和力的RNA。所有这些都能够支持大肠杆菌 RNase H 切割它们所针对的 HSV mRNA。同源物在含有 10% 胎牛血清 (FCS)、HeLa 胞质提取物、HeLa 核提取物的细胞培养基中以及完整 HeLa 细胞中的稳定性表明，ISIS 1080 在 48 小时内最能抵抗溶核消化。与完全硫代寡核苷酸相比，部分PS寡核苷酸通过FCS中的核酸外切酶活性和细胞提取物或完整细胞中的核酸内切酶活性表现出增强的降解。因此，与完全 PS 寡核苷酸相比，HeLa 细胞中部分 PS 寡核苷酸针对 HSV-1 的功效降低可能是由于混合 PO/PS 寡核苷酸的降解增加所致。
Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5'(ISIS 1370), both the 3' and 5'(ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNase H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.