Vosberg H P, Eckstein F
Biochemistry. 1977 Aug 9;16(16):3633-40. doi: 10.1021/bi00635a020.
We have synthesized fd and phi X174DNA in the presence of 2'-deoxyadenosine 5'-O-(1-thiotriphosphate) (dATP alpha S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized E. coli cells or crude cell extracts of E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP alpha S suggests that the dAMP has been completely substituted by 2'-deoxyadenosine 5'-0-phosphorothioate (dAMPS). The substituted DNAs have the same sedimentation coefficients, similar buoyant density, infectivity, and thermal stability as the unsubstituted DNAs. The procedure therefore allows specific modification at the 5' position of dA, dC, or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analogue of poly[r(A-U)] (Strothkamp, K.G., and Lippard, S.J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 2536), the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by electron microscopy.
我们使用经乙醚通透处理的大肠杆菌细胞或大肠杆菌DNA聚合酶I的粗细胞提取物，在2'-脱氧腺苷5'-O-(1-硫代三磷酸酯)(dATPαS)以及dCTP和dTTP的相应硫代磷酸酯衍生物存在的情况下，合成了fd和phi X174 DNA。在硫代磷酸酯存在的情况下，参与DNA形成或分解的酶的反应速率会降低。用[35S]dATPαS掺入的标记量表明，dAMP已被2'-脱氧腺苷5'-0-硫代磷酸酯(dAMPS)完全取代。取代后的DNA与未取代的DNA具有相同的沉降系数、相似的浮力密度、感染性和热稳定性。因此，该方法允许在DNA中dA、dC或dT的5'位置进行特异性修饰。鉴于最近已证明Pt2+配合物与聚[r(A-U)]的硫代磷酸酯类似物特异性结合(斯特罗斯坎普，K.G.，和利帕德，S.J.(1976年)，《美国国家科学院院刊》73，2536)，含硫代磷酸酯的DNA的合成可能可用于通过电子显微镜进行DNA测序。
