Unwinding of RNA G-Quadruplexes by DExH Helicases

The rG4 unwinding assays were performed using gel shift according to the procedure described in a published work in three independent repetitions (41 (link)). Briefly, different concentrations of purified DExH1 and DExH15 were mixed with 160 nM rG4 substrates in 25 mM Tris–HCl, pH 8.0, 50 mM KCl, 5 mM MgCl₂, 2 U/μl RNase inhibitor, 10% glycerol, and 1 mM DTT. The mixture was incubated for 10 min at 37 °C. Then, one equal volume of 4 mM ATP was added along with 1.6 μM traps to initiate reactions and incubated for 30 min at 37 °C. Then 5× stop buffer (125 mM EDTA and 50% glycerol (v/v)) and proteinase K (final concentration of 2 mg/ml) were added sequentially. After waiting for 10 min at 37 °C, the reaction products were electrophoresed on a 12% native PAGE. Finally, the gels were imaged using the Cy3 channel on a ChemiDoc MP Visualization System (BioRad) and analyzed in ImageJ (National Institutes of Health).