Quantification of HIV RT-Associated RNase H Activity

HIV RT-associated RNase H activity was measured as described [49 (link),50 (link)] (using the RNase H inhibitor RDS175911 as a control.) In a 100 µL reaction volume containing 50 mM Tris-HCl buffer with pH 7.8, 6 mM MgCl₂, 1 mM dithiothreitol (DTT), 80 mM KCl, 0.25 µM hybrid RNA/DNA 5′-GAUCUGAGCCUGGGAGCU-Fluorescin-3′ (HPLC, dry, QC: Mass Check) (available from Metabion (Planegg, Germany)) 5′-Dabcyl-AGCTCCCAGGCTCAGATC-3′ (HPLC, dry, QC: Mass Check), there were increasing concentrations of an inhibitor, whose dilution was made in water, and 20 ng of wt RT, according to a linear range of dose-response curve. The reaction mixture was incubated for 1 h at 37 °C, stopped by the addition of EDTA, and products were measured with a multilabel counter plate reader Victor 3 (Perkin Elmer model 1420-051 (Waltham, MA, USA)) equipped with filters for 490/528 nm (excitation/emission wavelength).