Nucleotide metabolism, which gives rise to changing dNTP concentrations in the cell, provides insight into the cell cycle in cancer biology and metabolic diseases. dNTP concentrations are highest during cell proliferation. However, the lack of a sensitive assay, and low concentrations of dNTPs in tissue, makes it challenging to study tissue dNTP pools, and thus the role of nucleotide metabolism is not well understood.
In a recent publication in Nucleic Acids Research, Purhonen et al. report the development and optimization of a fluorometric assay using EvaGreen® Dye to measure dNTP concentration in tissue. Measuring dNTP concentrations in tissue has traditionally relied on incorporating radioactive dNTPs or HPLC-MS instrumentation which is not accessible to many researchers. Fluorimetric assays have had limitations, such as lower sensitivity and inhibition of DNA polymerase by tissue extracts. In addition, the highly unbalanced dNTP concentrations needed to measure tissue levels of dNTPs with this technique may cause the incorporation of non-complimentary deoxyribonucleotides by the low fidelity of non-proofreading polymerase, resulting in high background fluorescence.
The authors optimized the conditions for measuring dNTP concentrations from tissue. The assay used a 197 nt designed template with a single dNTP detection site at the 3’ end and a high-fidelity Q5 DNA polymerase to minimize incorporation of noncomplimentary dNTPs. The background fluorescence remained nearly constant during the reaction time and produced a fluorescent signal proportional to the added limiting dNTP. The authors reported that the assay is sensitive enough to detect as little as 50 fmol of dNTP in mouse tissue samples and effectively overcomes the polymerase inhibition and background fluorescence which made previous fluorimetric assays less sensitive.
EvaGreen® Dye is highly specific for dsDNA and has a low fluorescence background due to its release on-demand mechanism. The dye exists in a quenched dimeric form that is released as a high affinity monomeric form once DNA is available. As a result, EvaGreen® Dye can be used at saturating concentrations without DNA polymerase inhibition. The fluorescence of EvaGreen® Dye is proportional to the molar concentration and length of dsDNA, making it an ideal dye for this assay.
The high specificity of EvaGreen® Dye for dsDNA also allows for the elimination of interfering rNTP signal using a thermostable RNAse HII and reading the fluorescence signal at a temperature at which the nicked DNA is denatured, but the intact product remains double-stranded. Cleavage products were verified by denaturing polyacrylamide gel electrophoresis and stained with PAGE GelRed®.
Besides being accessible to most research groups, this assay potentially offers other advantages. First, the quantification reaction occurs in a single microplate and requires only pipetting of a reagent mix and the sample, thus eliminating additional steps that introduce error. Secondly, a typical qPCR instrument can be used for temperature control and to read the fluorescence.
Purhonen, J., Banerjee, R., McDonald, A.E., Fellman, V., Kallijärvi, J., A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase. Nucleic Acids Research 48, e87 (2020). https://doi.org/10.1093/nar/gkaa516