| Assay Method Information | |
| | RNAP-Inhibition Assay |
| Description: | Fluorescence-detected RNAP inhibition assays in Tables 1 and 4 were performed using a DNA fragment containing the lacUV5 promoter as a template and the profluorescent ATP analog γ-[2′-(2-benzothiazoyl)-6′-hydroxybenzothiazole]-ATP(BBT-ATP, Jena Biosciences) as a substrate (methods as in ref 32). Fluorescence-detected RNAP inhibition assays in Figure 2C were performed essentially as described in refs 33 and 34. The assay used a proprietary circular single-stranded DNA as a template (Kool NC-45 Universal RNAP Template, Epicenter). The synthesized RNA was quantified using RiboGreen (ThermoFisher), which exhibits fluorescence enhancement upon binding to RNA. A volume of 0.2 μL of MRL-436 or Rif [10 concentrations from a 3-fold serial dilution in 100% DMSO (v/v)] was loaded into a 384-well plate using an Echo liquid handler (Labcyte). A total of 5 μL of 20 nM recombinant RNAP in assay buffer [50 mM HEPES (pH = 8.0), 50 mM KCl, 4 mM MgCl2, 5% glycerol (v/v), 1 mM DTT, 0.1 mg mL−1 BSA, and 0.01% Triton X-100 (v/v)] was then added. After a 30 min incubation at RT, 5 μL of assay buffer solution containing 0.04 ng/μL DNA template and rNTPs (ATP, CTP, GTP, UTP; 600 μM each; Lucigen) was added to start the reaction. After incubation for 2 h at 37 °C, 70 μL of Quant-iT RiboGreen RNA Reagent [1:350 dilution in buffer containing 10 mM Tris-HCl (pH = 7.5) and 1 mM EDTA] was added to stop the reaction and develop the signal. The plate was read using a PheraStar Plus plate reader (BMG). |
| Affinity data for this assay | |
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