Biochemical and physicochemical properties of phosphorodithioate DNA
Biochemical and physicochemical properties of phosphorodithioate DNA
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DOI：
10.1021/bi960318x
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发表时间：
1996-07-02
期刊：
影响因子：
2.9
通讯作者：
Caruthers, MH
中科院分区：
文献类型：
--
作者：
Cummins, L;Graff, D;Caruthers, MH
评价了含有各种构型的二硫代磷酸酯键的DNA寡聚体的生化和物理化学性质。双链体的稳定性研究，这是通过热变性分析与互补的未修饰的DNA，表明一个高度合作的过程类似于完全未修饰的双链体。发现含有二硫代磷酸酯键的低聚物相对于未修饰的双链体具有降低的解链温度，T-m抑制的程度与低聚物的二硫代磷酸酯组成百分比平行。相对于RNase H的活化，含有高达50%的二硫代磷酸酯键的DNA寡聚体能够以与未修饰的DNA相同的效率指导RNase H降解，而含有50至100%的DNA寡聚体以稍微降低的效率起作用。在极限浓度下，含有交替的二硫代磷酸酯和磷酸酯键的寡聚体能够在延长的孵育中直接RNA酶H降解靶RNA，而未修饰的寡聚体则不能。在HeLa细胞核和细胞质提取物、人血清中以及用核酸酶SI和DNase I评价含二硫代磷酸酯的寡聚体的核酸酶抗性。含有交替的二硫代磷酸酯和磷酸盐的低聚物在所有系统中对降解具有高度抗性。然而，具有一个以上未修饰的键分离二硫代磷酸酯的寡聚体被DNA酶I快速降解，同时在所有其他测试系统中表现出降解稳定性。这些结果表明，含二硫代磷酸酯的DNA寡聚体具有高度的核酸酶抗性，能够与互补核酸序列形成稳定的双链体，并有效地指导靶RNA的RNA酶H降解。
The biochemical and physicochemical properties of DNA oligomers containing phosphorodithioate linkages in various configurations were evaluated. Duplex stability studies, which were carried out by thermal denaturation analysis with complementary unmodified DNA, indicated a highly cooperative process similar to completely unmodified duplexes. Oligomers containing phosphorodithioate linkages were found to have reduced melting temperatures relative to unmodified duplexes, with the degree of T-m depression paralleling the percent phosphorodithioate composition of the oligomer. Relative to activation of RNase H, DNA oligomers containing up to 50% phosphorodithioate linkages were able to direct RNase H degradation with the same efficiency as unmodified DNA while those containing from 50 to 100% acted with somewhat reduced efficiency. At Limiting concentrations, an oligomer containing alternating phosphorodithioate and phosphate linkages was able to direct RNase H degradation of the target RNA in an extended incubation, while an unmodified oligomer did not. The nuclease resistance of phosphorodithioate-containing oligomers was evaluated in HeLa cell nuclear and cytoplasmic extracts, in human serum, and with nucleases SI and DNase I. Oligomers containing alternating phosphorodithioate and phosphate were highly resistant to degradation in all systems. However, oligomers having more than one unmodified linkage separating phosphorodithioates were degraded rapidly by DNase I, while demonstrating stability to degradation in all other systems tested. These results indicate that phosphorodithioate-containing DNA oligomers are highly nuclease-resistant, are able to form stable duplexes with complementary nucleic acid sequences, and efficiently direct RNase H degradation of target RNA.