Monia B P, Johnston J F, Sasmor H, Cummins L L
Department of Molecular Pharmacology and Division of Medicinal Chemistry, Isis Pharmaceuticals, Carlsbad, California 92008, USA.
J Biol Chem. 1996 Jun 14;271(24):14533-40. doi: 10.1074/jbc.271.24.14533.
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.
我们之前曾描述过针对人Ha-ras的17聚体均匀硫代磷酸酯反义序列的构效关系研究。为了进一步改善反义寡核苷酸的药理特性，对该17聚体序列的构效关系研究进行了扩展，以考察用磷酸二酯键取代硫代磷酸酯主链连接键的效果，以及在硫代磷酸酯和磷酸二酯寡核苷酸中引入各种2'-糖修饰对寡核苷酸体外抗核酸酶稳定性和细胞内反义活性的影响。用磷酸二酯键取代三个或更多硫代磷酸酯连接键会极大地损害核酸酶抗性和反义活性，且这些效应与寡核苷酸中掺入的磷酸二酯键数量直接相关。然而，通过掺入适当的2'-烷氧基糖修饰，磷酸二酯寡核苷酸获得了足以在细胞中产生有效反义效应的显著核酸酶抗性。这些糖修饰在磷酸二酯主链中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，对核酸酶降解具有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持续时间。
