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| | FRET-Based RNase H Assay |
| Description: | RNaseH assays were performed using an 18-nucleotide 3-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5-dabcyl conjugated DNA. The increase in fluorescence as a result of RNase H hydrolysis was monitored with a Spectramax Gemini EM fluorescence spectrometer. Data were analyzed using the instrument manufacturer SoftMax pro software. To determine IC50 values, the slope of the curve representing the time dependent increase in fluorescence was determined at 10 min, when initial rate conditions are met and substrate depletion is not significant. The slope values were plotted against the logarithm of the inhibitor concentrations, and IC50 values were determined using SigmaPlot software. The two previously characterized RNase H inhibitors beta-thujaplicinol and manicol were used as positive controls, yielding IC50 values in agreement with previously published data (250 +/- 23 nM for thujaplicinol and 0.6 +/- 0.09 uM for manicol). |
| Affinity data for this assay | |
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