Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 119991 Moscow, Russia.
Nucleic Acids Res. 2018 Jul 6;46(12):e73. doi: 10.1093/nar/gky247.
To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3- and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3' ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.
为了开发与 Taq DNA 聚合酶活性兼容的 dNTP 结构修饰物，我们合成了 8 种与 Cy3 或 Cy5 染料类似物缀合的 dUTP 衍生物，这些类似物在整个荧光团中在电荷和电荷分布上有所不同。这些 dUTP 衍生物以及商业的 Cy3 和 Cy5-dUTP 被用于 Taq 聚合酶依赖性聚合酶链反应（PCR）以及使用含有一个、两个和三个相邻腺嘌呤核苷酸的模型模板的引物延伸反应中进行了研究。使用荧光测量法估计了扩增 DNA 的相对量以及表征标记的 dUMP 掺入的动力学参数 Km 和 Vmax，并进行了分析。用带等电两性离子的 Cy3 或 Cy5 荧光染料标记的 dUTP 被 Taq 聚合酶有效利用的程度大约比用带负电荷的 Cy3 或 Cy5 类似物标记的 dUTP 高一个数量级。还观察到 Taq 聚合酶的核苷酸转移酶活性，导致将带有等电或正电荷的荧光染料标记的 dUMP 添加到 DNA 的 3' 末端。在荧光团或与 dNTP 缀合的其他官能团中引入相互补偿的电荷可以被认为是创建与 PCR 兼容的修饰核苷三磷酸的基础。
