RNA Helix-Destabilizing Assays with σNS

RNA helix-destabilizing assays were conducted as described^{38 (link)} with modifications. Briefly, various concentrations of σNS, σNS-R6A, and σNS-ΔN17 (2.5, 5, 10, and 20 µM) and 0.5 μM dsRNA helix substrate were incubated at 37 °C for 1 h in a reaction volume of 20 μL containing 25 mM HEPES-KOH (pH 8.0), 100 mM NaCl, 2 mM MgCl₂, 2 mM DTT, and 5 U RNase-OUT (Invitrogen). Reactions were terminated by the addition of 5 U proteinase K (NEB) for 15 min and 2.5 μL 5× loading buffer (100 mM Tris-HCl (pH 7.5), 50% glycerol, and bromophenol blue). The samples were electrophoresed in 4–20% native polyacrylamide gels (Genscript USA), and gels were scanned using a Typhoon 9200 imager (GE Healthcare). Bacterial-expressed maltose binding protein (MBP; 20 μM) and norovirus p41 protein (20 μM) were used as negative and positive controls, respectively. The effect of bile acids on σNS RNA helix-destabilizing activity was determined by incubating 20 and 40 μM of different components of bile acid derivatives, including TASAH, SGAH, CHAPS, and CHAPSO, with 20 μM σNS for 1 h before initiation of the helix-destabilization assay. Experiments were conducted three times.