Agrawal S, Mayrand S H, Zamecnik P C, Pederson T
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Proc Natl Acad Sci U S A. 1990 Feb;87(4):1401-5. doi: 10.1073/pnas.87.4.1401.
Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA. Oligodeoxynucleotides complementary to nucleotides 2-14 of human U1 small nuclear RNA were investigated. Extensive cleavage of U1 RNA was observed with the unmodified oligodeoxynucleotide and with the phosphorothioate analogue but not with U1-complementary oligodeoxynucleotides containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate internucleoside linkages. Additional experiments using a 514-nucleotide-long RNA substrate demonstrated the capacity of complementary phosphodiester- and phosphorothioate-linked oligodeoxynucleotides (but not ones containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate linkages) to serve as RNase H targets when hybridized to an internal RNA site. Detailed comparisons revealed phosphodiester-linked oligodeoxynucleotides to be more efficient than the comparable phosphorothioate-linked oligomers with respect to RNase H action. Various pentadecamer oligodeoxynucleotides complementary to the 514-nucleotide-long test RNA and containing 2-6 consecutive phosphodiester- or phosphorothioate-linked nucleotides flanked by RNase H-resistant methylphosphonate linkages afforded precise "site-directed" RNase H excision within the DNA.RNA hybrid. These results serve to assort modified oligodeoxynucleotide-containing hybrids into RNase H-sensitive and -resistant classes and also provide clues as to how RNase H makes contact with the DNA strand in a DNA.RNA hybrid.
对含有磷酸二酯键或修饰的核苷间连接的寡脱氧核苷酸进行了研究，考察它们在与RNA中的互补序列杂交后，被HeLa细胞核提取物中存在的核糖核酸酶H活性作用的能力。研究了与人U1小核RNA核苷酸2 - 14互补的寡脱氧核苷酸。未修饰的寡脱氧核苷酸和硫代磷酸酯类似物能使U1 RNA发生广泛切割，而含有甲基膦酸酯、吗啉代磷酸酯或丁基酰胺代磷酸酯核苷间连接的U1互补寡脱氧核苷酸则不能。使用一个514个核苷酸长的RNA底物进行的额外实验表明，互补的磷酸二酯键连接和硫代磷酸酯键连接的寡脱氧核苷酸（但不包括含有甲基膦酸酯、吗啉代磷酸酯或丁基酰胺代磷酸酯连接的寡脱氧核苷酸）在与内部RNA位点杂交时能够作为核糖核酸酶H的作用靶点。详细比较表明，就核糖核酸酶H的作用而言，磷酸二酯键连接的寡脱氧核苷酸比类似的硫代磷酸酯键连接的寡聚物更有效。各种与514个核苷酸长的测试RNA互补且含有2 - 6个连续的磷酸二酯键或硫代磷酸酯键连接的核苷酸、两侧为对核糖核酸酶H有抗性的甲基膦酸酯连接的十五聚体寡脱氧核苷酸，能在DNA - RNA杂交体中实现精确的“定点”核糖核酸酶H切割。这些结果有助于将含有修饰寡脱氧核苷酸的杂交体分为对核糖核酸酶H敏感和抗性两类，也为核糖核酸酶H如何与DNA - RNA杂交体中的DNA链接触提供了线索。
