Quartin R S, Brakel C L, Wetmur J G
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029.
Nucleic Acids Res. 1989 Sep 25;17(18):7253-62. doi: 10.1093/nar/17.18.7253.
Oligodeoxynucleotides with different arrangements of methylphosphonate linkages were examined for nuclease sensitivity in vitro, stability in tissue culture, and ability to form RNase H-sensitive substrates with complementary RNA. After nuclease treatment, resistance was demonstrated by the ability to alter the electrophoretic mobility of a labeled complementary phosphodiester oligodeoxynucleotide. Both 5'- and 3'-exonuclease activities were retarded by methylphosphonate linkages. Methylphosphonate-containing oligodeoxynucleotides with 1-5 adjacent phosphodiester linkages were tested as substrates for the endonucleases DNase I and DNase II. The results indicated that a span of three or fewer contiguous internal phosphodiester linkages led to the greatest resistance to endonuclease. However, in serum-supplemented culture medium half-lives of these oligodeoxynucleotides were independent of the number of contiguous phosphodiester linkages. Methylphosphonate-containing oligodeoxynucleotides were hybridized to RNA runoff transcripts and tested as substrates for RNase H. The results indicated that a span of three internal phosphodiester linkages in the oligodeoxynucleotide was necessary and sufficient to direct cleavage of the RNA in the duplex.
研究了具有不同甲基膦酸酯键排列的寡脱氧核苷酸在体外对核酸酶的敏感性、在组织培养中的稳定性以及与互补RNA形成对核糖核酸酶H敏感底物的能力。核酸酶处理后，通过改变标记的互补磷酸二酯寡脱氧核苷酸的电泳迁移率来证明其抗性。5'-和3'-外切核酸酶活性均受到甲基膦酸酯键的抑制。测试了具有1-5个相邻磷酸二酯键的含甲基膦酸酯的寡脱氧核苷酸作为核酸内切酶DNase I和DNase II的底物。结果表明，三个或更少连续内部磷酸二酯键的跨度导致对核酸内切酶的最大抗性。然而，在补充血清的培养基中，这些寡脱氧核苷酸的半衰期与连续磷酸二酯键的数量无关。将含甲基膦酸酯的寡脱氧核苷酸与RNA径流转录本杂交，并作为核糖核酸酶H的底物进行测试。结果表明，寡脱氧核苷酸中三个内部磷酸二酯键的跨度对于指导双链体中RNA的切割是必要且充分的。
