Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′-(α-P-seleno)triphosphate and DNA polymerase for X-ray crystallography via MAD
Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′-(α-P-seleno)triphosphate and DNA polymerase for X-ray crystallography via MAD
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DOI：
10.1021/ja0383221
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发表时间：
2004-01-21
影响因子：
15
通讯作者：
Huang, Z
中科院分区：
文献类型：
--
作者：
Carrasco, N;Huang, Z
本文首次报道了利用两种α-Se-TTP非对映体（Sp和Rp）和DNA聚合酶合成两种硒酸磷（PSe） DNA的研究。实验结果表明，Klenow在与天然TTP相同的水平上对这两种非对映体具有同样的识别能力。对外切酶III的酶切抗性证实了PSe基团在预期位点的结合，DNA I和DNA II的不同酶切抗性模式表明PSe中心（Sp或Rp）的构型差异。与化学合成不同的是，化学合成仅限于短DNA，并且需要分离PSe DNA非对映体，而这种酶促方法可以用于制备更长的DNA，而无需分离非对映体。这种定量酶法对于大规模合成具有多个PSe基团的较长dna特别有价值，因为它们的x射线晶体结构可以通过MAD相控技术确定。
We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two α-Se-TTP diastereomers (Sp and Rp) and DNA polymerase. The experimental results indicate that Klenow equally recognizes the two individual diastereomers at the same level as natural TTP. The incorporations of the PSe groups at the expected sites have been confirmed by the digestion resistance to exonuclease III, and the different patterns of the digestion resistance of DNA I and II indicate the configurational differences of the PSe centers (Sp or Rp). Unlike chemical synthesis, which is limited to short DNAs and where the separation of the PSe DNA diastereomers is necessary, this enzymatic method can be used to prepare longer DNAs without diastereomer separation. This quantitative enzymatic approach is particular valuable for the synthesis of longer DNAs with multiple PSe groups in large scale for their X-ray crystal structure determination by the MAD phasing technique.