Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates
Synthesis and enzymatic incorporation of modified deoxyuridine triphosphates
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DOI：
10.1039/b906956a
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发表时间：
2009-01-01
影响因子：
3.2
通讯作者：
Howorka, Stefan
中科院分区：
文献类型：
--
作者：
Borsenberger, Vinciane;Kukwikila, Mikiembo;Howorka, Stefan
我们描述了在嘧啶基碱基的位置5位置5的2'-脱氧尿苷-5'-三磷酸衍生物的启动器，具有变化的长度，散装和柔韧性。与末端官能团的核苷酸类似物由于其在DNA链的功能标记中的应用潜力而引起了人们的关注。在合成核苷酸类似物的过程中，优化了Yoshikawa磷酸化程序的方法，从而导致了一种减少副产品量的方法，并且与基础上附着的不稳定官能团兼容。使用两种不同的DNA聚合酶系统地研究了连接器组成对酶促掺入DNA中的影响。来自B-聚合酶家族的深通风口（R）EXO（ - ）接受了大多数核苷酸类似物作为底物，而来自A-family的TAQ熟练少一些。两种聚合酶都有掺入5-（3-氨基 - 丙型-1-ynyl）-2'-脱氧尿苷三磷酸的困难。聚合酶活性位点的分子模型用于合理化为什么该核苷酸不被接受为底物。
We describe the synthesis of 2'-deoxyuridine-5'-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep Vent(R) exo(-) from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2'-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.