Abstract
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.
Highlights
The levels of the four deoxynucleoside triphosphates are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis
After termination of the reaction, streptavidin Sepharose was added to pull-down the oligonucleotides, which were denatured with 0.1N NaOH and washed extensively to remove free EdUTP and template
The current method used one common biotin-labeled primer annealed with four different templates, each specific for dATP, dTTP, dGTP and dCTP
Summary
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. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. Several methods have been developed to quantitate cellular levels of the four dNTPs. Among them, enzymatic- and liquid chromatography-based assays have been widely used. The disadvantage of this technique has been the handling storage and disposal of radioactive isotopes in www.nature.com/scientificreports line with government or institute regulations To solve this issue, dual-quenched fluorophore-labeled oligonucleotides for dNTP quantification were developed[18]. The method was adapted to a microplate assay in order to increase analysis capacity We used this assay to demonstrate that dNTP levels are modified in response to different chemotherapeutic agents
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