Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras
Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras
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DOI：
10.1074/jbc.271.24.14533
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发表时间：
1996-06-14
影响因子：
4.8
通讯作者：
Cummins, LL
中科院分区：
文献类型：
--
作者：
Monia, BP;Johnston, JF;Cummins, LL
我们先前已经描述了针对人Ha-ras的17-mer均匀硫代磷酸酯反义序列的结构-活性研究。为了进一步改善反义寡核苷酸的药理学性质，对该17聚体序列的结构-活性研究进行了扩展，以检查用磷酸二酯键取代硫代磷酸酯骨架键的影响以及将各种2 '-糖修饰掺入硫代磷酸酯和磷酸二酯寡核苷酸对体外寡核苷酸对核酸酶的稳定性和对细胞中反义活性的影响。用磷酸二酯键取代三个或更多个硫代磷酸酯键大大损害了核酸酶抗性和反义活性，并且这些效应与掺入寡核苷酸中的磷酸二酯键的数量直接相关。然而，通过掺入适当的2 '-烷氧基糖修饰，赋予磷酸二酯寡核苷酸实质性的核酸酶抗性，足以在细胞中获得有效的反义作用。由磷酸二酯主链中的这些糖修饰赋予的核酸酶稳定性和反义活性与2 '-烷氧基取代基的大小相关（戊氧基>丙氧基>甲氧基>脱氧）。此外，由对溶核降解表现出部分抗性的寡核苷酸介导的反义活性取决于寡核苷酸浓度和寡核苷酸处理的持续时间。
We have previously described structure-activity studies on a 17-mer uniform phosphorothioate antisense sequence targeted to human Ha-ras. In an effort to further improve the pharmacological properties of antisense oligonucleotides, structure-activity studies on this 17-mer sequence were expanded to examine both the effects of replacing phosphorothioate backbone linkages with phosphodiester linkages and the effects of incorporating various 2'-sugar modifications into phosphorothioate and phosphodiester oligonucleotides on oligonucleotide stability against nucleases in vitro and on antisense activity in cells. Replacement of three or more phosphorothioate Linkages with phosphodiester linkages greatly compromised both nuclease resistance and antisense activity, and these effects correlated directly with the number of phosphodiester linkages incorporated into the oligonucleotide. However, substantial nuclease resistance, sufficient for obtaining potent antisense effects in cells, was conferred to phosphodiester oligonucleotides by incorporation of appropriate 2'-alkoxy sugar modifications. Nuclease stability and antisense activity imparted by these sugar modifications in phosphodiester backbones correlated with the size of the 2'-alkoxy substituent (pentoxy > propoxy > methoxy > deoxy). Furthermore, antisense activity mediated by oligonucleotides that exhibit partial resistance to nucleolytic degradation was dependent on both oligonucleotide concentration and the duration of oligonucleotide treatment.