Mass check

HIV RT-associated RNase H activity was measured as described (Corona et al., 2016b (link)) in a 100-µl reaction volume containing 50 mM of Tris-HCl buffer, pH 7.8, 6 mM of MgCl₂, 1 mM of dithiothreitol (DTT), 80 mM of KCl, 0.25 µM of hybrid RNA/DNA 5′-GAUCUGAGCCUGGGAGCU-Fluorescin-3′ (high-performance liquid chromatography (HPLC), dry, QC: Mass Check) (available from Metabion) 5′-Dabcyl-AGCTCCCAGGCTCAGATC-3′ (HPLC, dry, QC: Mass Check), increasing concentrations of inhibitors, whose dilution were made in water, and different amounts of enzymes according to a linear range of the dose–response curve. The reaction mixture was incubated for 1 h at 37°C and stopped by the addition of EDTA, and products were measured with a multilabel counter plate reader Victor 3 equipped with filters for 490/528 nm.

HIV-1 RT group M subtype B was expressed and purified as described [49 (link)]. The HIV-1 RT-associated RNase H inhibition assay was performed as described previously [49 (link),50 (link)]. Briefly, anti-RNase H activity was measured in 100 µL reaction volume containing 50 mM Tris-HCl buffer pH 7.8, 6 mM MgCl₂, 1 mM dithiothreitol (DTT), 80 mM KCl, and HIV-1 RT. Reactions were started by addition of hybrid RNA/DNA 5′-GAUCUGAGCCUGGGAGCU-fluorescein-3′ (high-performance liquid chromatography [HPLC], dry, QC: Mass Check) (available from Metabion, Planegg, Germany) and 5′-dabcyl-AGCTCCCAGGCTCAGATC-3′ (HPLC, dry, QC: Mass Check) at a final concentration of 0.25 µM. The reaction mixtures were incubated in a multilabel counter plate reader Victor 3 (Model 1420-051, Perkin Elmer, Waltham, MA, USA) for 10 min at 37 °C, followed by product quantification at 490/528 nm (excitation/emission wavelength). All experiments were performed in triplicate.