Latimer L J, Hampel K, Lee J S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Nucleic Acids Res. 1989 Feb 25;17(4):1549-61. doi: 10.1093/nar/17.4.1549.
More than twenty repeating sequence DNAs containing phosphorothioates were prepared from the appropriate dXTPs with DNA polymerase I. The Tms of the modified DNAs were all lower than the parent polymers. A phosphorothioate group 5' to a pyrimidine gave rise to a large decrease than 5' to a purine, e.g., poly(dA).poly(dT) = 50 degrees; poly(dsA).poly(dT) = 44 degrees; poly(dA).poly(dsT) = 33 degrees; and poly(dsA).poly(dsT) = 26 degrees. The presence of phosphorothioate groups had a dramatic effect on triplex formation; poly[d(TC)].poly[d(sGsA)] spontaneously dismutases to a triplex at pH 8 whereas triplex formation in poly[d(sTsC)].poly[d(GA)] was inhibited. Surprisingly poly(dsG).poly(dC) had a Tm which initially decreased with increasing ionic strength. Resistance to digestion with pancreatic DNAse I did not correlate with phosphorothioate content. Poly[d(AsT)], poly[d(TsC)].poly[d(sGA)] and poly[d(sTG)].poly[d(sCA)] were resistant whereas poly[d(sAT)] and poly[d(sTsTG)].poly[d(CsAsA)] were rapidly degraded. Thus phosphorothioate groups cause small conformational changes and may reveal new families of conformational polymorphisms.
使用DNA聚合酶I从适当的dXTPs制备了二十多种含硫代磷酸酯的重复序列DNA。修饰后的DNA的熔解温度均低于亲本聚合物。嘧啶5'端的硫代磷酸酯基团比嘌呤5'端的硫代磷酸酯基团导致的熔解温度降低幅度更大，例如，聚（dA）·聚（dT）=50℃；聚（dsA）·聚（dT）=44℃；聚（dA）·聚（dsT）=33℃；聚（dsA）·聚（dsT）=26℃。硫代磷酸酯基团的存在对三链体形成有显著影响；聚[d（TC）]·聚[d（sGsA）]在pH 8时自发歧化为三链体，而聚[d（sTsC）]·聚[d（GA）]中的三链体形成受到抑制。令人惊讶的是，聚（dsG）·聚（dC）的熔解温度最初随离子强度增加而降低。对胰DNA酶I消化的抗性与硫代磷酸酯含量无关。聚[d（AsT）]、聚[d（TsC）]·聚[d（sGA）]和聚[d（sTG）]·聚[d（sCA）]具有抗性，而聚[d（sAT）]和聚[d（sTsTG）]·聚[d（CsAsA）]则迅速降解。因此，硫代磷酸酯基团会引起小的构象变化，并可能揭示新的构象多态性家族。
