Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR
Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR
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DOI：
10.1093/nar/29.9.1898
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发表时间：
2001-05-01
影响因子：
14.9
通讯作者：
Williams, DM
中科院分区：
文献类型：
--
作者：
Gourlain, T;Sidorov, A;Williams, DM
将潜在的催化基团掺入DNA中对于新型脱氧核酶的体外选择是有意义的。我们已经设计了一系列的合成路线，以C7修饰的7-deaza-dATP衍生物与侧氨基丙基，Z-氨基丙烯基和氨基丙炔基侧链。这些修饰的三磷酸盐已在PCR期间作为Tao聚合酶的底物进行了测试。所有的修饰都能被这种酶所耐受，其中氨基丙炔基侧链得到最好的结果。大多数蛋白质酶在其活性部位具有一种以上类型的催化基团。通过使用C5-咪唑基修饰的dUTP与3-（氨基丙炔基）-7-deaza-dATP一起代替天然核苷酸dTTP和dATP，我们已经证明了在PCR期间氨基和咪唑基部分同时掺入DNA分子中。含有四种天然碱基的PCR产物被XbaI完全消化，而含有修饰的7-脱氮-dATP类似物的PCR产物不被切割。使用质谱法已经获得了咪唑修饰的dUTP和氨基修饰的7-deaza-dATP在PCR期间同时掺入的直接证据。
The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Tao polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by Xbal, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.