Journal
ANALYTICAL CHEMISTRY
Volume 91, Issue 22, Pages 14561-14568Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.9b03624
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Funding
- French National Research Agency (Investissements d'Avenir project Labex NanoSaclay) [ANR-10-LABX-0035]
- French National Research Agency (ANR project AMPLIFY)
- Institut Universitaire de France (IUF)
- French Institut National Du Cancer
- Direction Generale de l'Offre de Soins (INCa) [PRTk 16158 Gynomir]
- Direction Generale de l'Offre de Soins (DGOS) [PRTk 16158 Gynomir]
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The quantification of cellular deoxyribonucleo-side triphosphate (dNTP) levels is important for studying pathologies, genome integrity, DNA repair, and the efficacy of pharmacological drug treatments. Current standard methods, such as enzymatic assays or high-performance liquid chromatography, are complicated, costly, and labor-intensive, and alternative techniques that simplify dNTP quantification would present very useful complementary approaches. Here, we present a dNTP assay based on isothermal rolling circle amplification (RCA) and rapid time-gated Forster resonance energy transfer (TG-FRET), which used a commercial clinical plate reader system. Despite the relatively simple assay format, limits of detection down to a few picomoles of and excellent specificity for each dNTP against the other dNTPs, rNTPs, and dUTP evidenced the strong performance of the assay. Direct applicability of RCA-FRET to applied nucleic acid research was demonstrated by quantifying all dNTPs in CEM-SS leukemia cells with and without hydroxyurea or auranofin treatment. Both pharmacological agents could reduce the dNTP production in a time- and dose-dependent manner. RCA-FRET provides simple, rapid, sensitive, and specific quantification of intracellular dNTPs and has the potential to become an advanced tool for both fundamental and applied dNTP research.
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