使用胸苷5'-（α-P-硒代）三磷酸和DNA聚合酶通过多波长反常散射法进行X射线晶体学分析的磷硒酸酯DNA的酶促合成。

Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5'-(alpha-P-seleno)triphosphate and DNA polymerase for X-ray crystallography via MAD.

作者信息

Carrasco Nicolas, Huang Zhen

机构信息

Department of Chemistry, Brooklyn College, and Program of Biochemistry and Chemistry, The Graduate School, The City University of New York, 2900 Bedford Avenue, Brooklyn, New York 11210, USA.

出版信息

J Am Chem Soc. 2004 Jan 21;126(2):448-9. doi: 10.1021/ja0383221.

DOI:10.1021/ja0383221

PMID:14719925

Abstract

We report here the first study of enzymatic synthesis of two phosphoroselenoate (PSe) DNAs using the two alpha-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.

摘要

我们在此报告了第一项关于使用两种α-硒代胸苷三磷酸（α-Se-TTP）非对映异构体（Sp和Rp）以及DNA聚合酶酶促合成两种磷硒酸酯（PSe）DNA的研究。实验结果表明，Klenow对这两种单独的非对映异构体的识别程度与天然TTP相同。通过对外切核酸酶III的消化抗性已证实了PSe基团在预期位点的掺入，并且DNA I和II不同的消化抗性模式表明了PSe中心（Sp或Rp）的构型差异。与仅限于短DNA且需要分离PSe DNA非对映异构体的化学合成不同，这种酶促方法可用于制备更长的DNA而无需分离非对映异构体。这种定量酶促方法对于大规模合成具有多个PSe基团的更长DNA以通过MAD相位技术确定其X射线晶体结构特别有价值。