For the detection of RNA synthesis by ZIKV RdRp, we established a real-time assay based on the fluorescent dye SYTO 9, which binds dsRNA but not ssRNA template molecules. The fluorescence emitted was recorded in real-time using a Fluostar Optima fluorimeter (BMG Labtech) using excitation and emission filters at 485 and 520 nm, respectively. The assay records the synthesis of dsRNA in a reaction using a poly-U molecule as a template and ATP as the nucleotide substrate. This technique has been adapted from methods previously documented for the detection of DNA synthesis38 (link).
Reactions were performed in individual wells of black 96-well flat-bottom plates. The standard reaction contained 50 mM Tris-HCl, pH 7.5, 2.5 mM MnCl2, 500 μM ATP, 20 μg/mL poly-U, 0.1 mg/mL BSA and 0.25 μM SYTO 9 (50 μM stock solution in TE buffer pH 7.5). The assay was initiated by the addition of 250 nM ZIKV RdRp and the fluorescence was recorded over 30 min at 30 °C.
Variations on this assay, for example, different concentrations of reagents and/or the presence of additional compounds, are specifically indicated in each corresponding section. For graphical representation, background fluorescence obtained at time point 0 was subtracted from each value.
To determine Km and Vmax constants for ZIKV RdRp binding to poly-U ssRNA, standard reactions were carried out in increasing concentrations of the template (0.5–50 μg/mL) in the presence of ATP at 500 μM. The kinetic parameters for ATP were obtained from assays in the presence of increasing concentrations of this nucleotide (200–2250 μM) and using 3 μg/mL of poly-U.
IC50 values were obtained from standard reactions carried out in the presence of 3 μg/mL poly-U and 1500 μM ATP, and increasing concentrations of each inhibitor.
End-point fluorometric reactions were performed in black 96-well black-flat bottom plates at 30 °C in the presence of the same reagents as described above, but in the absence of dye. The reactions were quenched at 60 min by adding in 25 mM EDTA to the samples. Either SYTO9 or SYBR Green II dye was then added to the sample (0.25 μM or 1x, respectively) and the mix reaction was incubated at room temperature for 5 min to allow the stabilization of RNA-dye complexes and fluorescence emission. To determine background fluorescence levels, a negative control was assayed in parallel, where the reaction was quenched before adding ZIKV RdRp. The quenched control reaction was incubated for 1 h at 30 °C, and then 0.25 μM SYTO9 or 1 × SYBR Green II, respectively, was added to the sample and fluorescence recorded as described above.
Reactions were performed in individual wells of black 96-well flat-bottom plates. The standard reaction contained 50 mM Tris-HCl, pH 7.5, 2.5 mM MnCl2, 500 μM ATP, 20 μg/mL poly-U, 0.1 mg/mL BSA and 0.25 μM SYTO 9 (50 μM stock solution in TE buffer pH 7.5). The assay was initiated by the addition of 250 nM ZIKV RdRp and the fluorescence was recorded over 30 min at 30 °C.
Variations on this assay, for example, different concentrations of reagents and/or the presence of additional compounds, are specifically indicated in each corresponding section. For graphical representation, background fluorescence obtained at time point 0 was subtracted from each value.
To determine Km and Vmax constants for ZIKV RdRp binding to poly-U ssRNA, standard reactions were carried out in increasing concentrations of the template (0.5–50 μg/mL) in the presence of ATP at 500 μM. The kinetic parameters for ATP were obtained from assays in the presence of increasing concentrations of this nucleotide (200–2250 μM) and using 3 μg/mL of poly-U.
IC50 values were obtained from standard reactions carried out in the presence of 3 μg/mL poly-U and 1500 μM ATP, and increasing concentrations of each inhibitor.
End-point fluorometric reactions were performed in black 96-well black-flat bottom plates at 30 °C in the presence of the same reagents as described above, but in the absence of dye. The reactions were quenched at 60 min by adding in 25 mM EDTA to the samples. Either SYTO9 or SYBR Green II dye was then added to the sample (0.25 μM or 1x, respectively) and the mix reaction was incubated at room temperature for 5 min to allow the stabilization of RNA-dye complexes and fluorescence emission. To determine background fluorescence levels, a negative control was assayed in parallel, where the reaction was quenched before adding ZIKV RdRp. The quenched control reaction was incubated for 1 h at 30 °C, and then 0.25 μM SYTO9 or 1 × SYBR Green II, respectively, was added to the sample and fluorescence recorded as described above.
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