| Assay Method Information | |
| | Dengue Polymerase Assay (DENVpol) |
| Description: | The enzyme activity of dengue virus NS5 polymerase domain (DENVpol, serotype 2, New Guinea C strain) was measured as an incorporation of tritiated NMP into acid-insoluble RNA products. DENVpol assay reactions contained 100 nM recombinant enzyme, 50 nM heteropolymeric RNA, about 0.5 μCi tritiated NTP, 0.33 μM of competing cold NTP, 40 mM HEPES (pH 7.5), 3 mM dithiothreitol, and 2 mM MgCl2. Standard reactions were incubated for 3 hours at 30° C., in the presence of increasing concentration of inhibitor. At the end of the reaction, RNA was precipitated with 10% TCA, and acid-insoluble RNA products were filtered on a size exclusion 96-well plate. After washing of the plate, scintillation liquid was added and radiolabeled RNA products were detected according to standard procedures with a Trilux Topcount scintillation counter. The compound concentration at which the enzyme-catalyzed rate was reduced by 50% (IC50) was calculated by fitting the data to a non-linear regression (sigmoidal). |
| Affinity data for this assay | |
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