Quantitation of deoxynucleoside triphosphates by click reactions
Journal
Scientific Reports
Journal Volume
10
Journal Issue
1
Date Issued
2020
Author(s)
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. ? 2020, The Author(s).
SDGs
Other Subjects
2'-deoxyadenosine triphosphate; 2'-deoxycytidine 5'-triphosphate; 5-carboxytetramethylrhodamine succinimidyl ester; 5-ethynyl-2'-deoxyuridylic acid; copper; deoxyadenosine phosphate; deoxycytidine phosphate; deoxyguanosine phosphate; deoxyguanosine triphosphate; deoxyribonucleotide; deoxyuridine phosphate; pyrimidine nucleotide; rhodamine; thymidine 5'-triphosphate; chemistry; click chemistry; cycloaddition; HCT 116 cell line; HEK293 cell line; human; K-562 cell line; procedures; staining; Click Chemistry; Copper; Cycloaddition Reaction; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; Deoxyuracil Nucleotides; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; Rhodamines; Staining and Labeling; Thymine Nucleotides
Publisher
Nature Research
Type
journal article