PSI-7409 tetrasodium

Human DNA polymerase α, β, or γ is assayed in a 10-μL reaction mixture containing 50 mM Tris (pH 7.5), 50 mM NaCl, 3 mU/μL activated calf thymus DNA, a 20 μM concentration of all four natural deoxynucleoside triphosphates, 4 μCi [α-³²P]dCTP, 5 mM MgCl₂, and increasing concentrations of PSI-7409 (up to 1 mM), D-ddFCTP, or aphidicolin. DNA polymerase α, β, or γ is added to the reaction mixture to give final concentrations of 20, 18, and, 50 μg/mL, respectively. All reactions are run at 37°C and quenched at 30 min by mixing with 1 μL of 0.5 M EDTA. The radiolabeled products are quantified. A nonlinear fit is performed to determine the IC₅₀. The activity of RNA polymerase II is determined in a 25-μL in vitro transcription reaction mixture containing 100 ng of cytomegalovirus (CMV) immediate-early promoter DNA, 400 μM ATP, CTP, and UTP, 16 μM GTP, 10 μCi [α-³²P]GTP, 3 mM MgCl₂, and various concentrations of PSI-7409 (up to 1 mM), 3'-dCTP, or α-amanitin in transcription buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 20% glycerol). All reactions are run at 30°C and quenched at 60 min by mixing with 125 μL of stop solution (0.3 M Tris-HCl [pH 7.4], 0.3 M sodium acetate, 0.5% SDS, 2 mM EDTA, and 3 μg/mL tRNA). The RNA product is purified. The resulting samples contains 12 μL and the same volume of gel loading dye (98% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) is added. The samples are heated at 90°C for 5 min and loaded onto a 6% polyacrylamide sequencing gel. After running, the gel is exposed to a phosphorscreen, and the product is visualized and quantified by using a phosphorimager^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.