Gourlain T, Sidorov A, Mignet N, Thorpe S J, Lee S E, Grasby J A, Williams D M
Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
Nucleic Acids Res. 2001 May 1;29(9):1898-905. doi: 10.1093/nar/29.9.1898.
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 Taq 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 XbaI, 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.
将潜在的催化基团引入DNA对于新型脱氧核酶的体外筛选具有重要意义。我们设计了一系列合成路线，用于合成带有侧链氨丙基、Z - 氨丙烯基和氨丙炔基的三种C7修饰的7 - 脱氮 - dATP衍生物。这些修饰的三磷酸酯已作为Taq聚合酶在PCR过程中的底物进行了测试。该酶对所有这些修饰都具有耐受性，其中氨丙炔基侧链的效果最佳。大多数蛋白质酶在其活性位点含有不止一种类型的催化基团。通过使用C5 - 咪唑基修饰的dUTP与3 -（氨丙炔基）- 7 - 脱氮 - dATP共同替代天然核苷酸dTTP和dATP，我们已经证明在PCR过程中氨基和咪唑基部分可同时掺入到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产物未被切割。使用质谱法已获得在PCR过程中咪唑修饰的dUTP和氨基修饰的7 - 脱氮 - dATP同时掺入的直接证据。
