Fluorous base-pairing effects in a DNA polymerase active site
Fluorous base-pairing effects in a DNA polymerase active site
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DOI：
10.1002/chem.200401151
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发表时间：
2005-05-06
影响因子：
4.3
通讯作者：
Kool, ET
中科院分区：
文献类型：
--
作者：
Lai, JS;Kool, ET
我们通过使用配对边缘全氟化的核苷酸类似物描述了 DNA 聚合酶活性位点的选择性“氟”效应。合成并研究了 DNA 碱基类似物 2,3,4,5-四氟苯 (B-F) 和 4,5,6,7-四氟吲哚 (I-F) 以及烃对照苯 (B) 和吲哚 (I) 的 5'-三磷酸脱氧核苷酸衍生物，作为 DNA 聚合酶 I Klenow 片段 (KF exo-) 的底物。修饰的核苷酸存在于 DNA 模板中，或者在单核苷酸插入的稳态动力学研究中以三磷酸核苷的形式提供。当与模板链中的非天然碱基相对时，疏水性三磷酸核苷的掺入效率比天然脱氧三磷酸核苷高两个数量级。嘌呤类氟化吲哚核苷酸 (I-F) 是四种疏水性类似物中插入效率最高的，最有效的掺入发生在嘧啶类四氟苯 (B-F) 对面。在所有情况下，多氟化碱基对的处理比类似的烃对更有效。对超出这些对的聚合酶延伸进行的初步测试表明，只有 B-F 碱基明显延伸；这种低效的延伸与最近发表的有关其他非极性碱基对的数据一致。这些结果表明疏水性、堆积和空间相互作用在聚合酶介导的缺乏氢键的 DNA 碱基对复制中的重要性。这些发现进一步表明，多氟芳族碱基增强的疏水性可用于设计新的选择性碱基对，这些碱基对与复制中使用的天然沃森-克里克对正交。
We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene (B-F) and 4,5,6,7-tetrafluoroindole (I-F), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide (I-F) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene (B-F). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the B-F base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.