The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells.

中文翻译：

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通过点击反应定量脱氧核苷三磷酸。

四种脱氧核苷三磷酸（dNTP）的水平在细胞中受到严格控制，因为不正确或不平衡的dNTP库可能导致生长缺陷和致癌作用。用治疗剂治疗癌细胞后，典型dNTPs水平的变化可能为评估药物反应和作用方式提供关键信息。放射性同位素标记酶法已普遍用于定量细胞dNTP水平。但是，这种方法的缺点是处理生物危害材料。在这里，我们描述了使用点击化学取代模板依赖的DNA聚合中的放射性同位素标记，以定量四种经典dNTP。针对dCTP，dTTP，dATP和dGTP测量设计了特定的低聚物，并且在聚合反应过程中引入5-乙炔基-dUTP或C8-炔烃-dCTP使得荧光团缀合在固定的寡核苷酸上。这四个反应在106个细胞中存在的dNTPs浓度范围内给出的线性相关系数> 0.99，而对细胞rNTPs的干扰很小。我们提供的证据表明，用这种方法产生的数据与放射性同位素标记的数据相当。此外，基于该技术的稳健微孔板检测的设计和利用证明了dNTP对癌细胞中不同化学治疗剂的响应的调节。在106个细胞中存在的dNTP浓度范围内为99，而对细胞rNTP的干扰很小。我们提供的证据表明，用这种方法产生的数据与放射性同位素标记的数据相当。此外，基于该技术的稳健微孔板检测的设计和利用证明了dNTP对癌细胞中不同化学治疗剂的响应的调节。在106个细胞中存在的dNTP浓度范围内为99，而对细胞rNTP的干扰很小。我们提供的证据表明，用这种方法产生的数据与放射性同位素标记的数据相当。此外，基于该技术的稳健微孔板测定的设计和利用证明了dNTP对癌细胞中不同化学治疗剂的响应的调节。