Quantification of G4R1 Binding Kinetics

We have previously described the isolation [38 (link),42 (link)] and quantification [40 (link)] of active recombinant G4R1 from Rosetta 2 cells. The apparent K_d was estimated using gel mobility shift assays (GMSA) as previously described [40 (link)]. Briefly, recombinant G4R1 at concentrations of 10–500 pM was incubated with 1 pM 5’-[32P]-end-labeled G4-DNA in Res buffer (50 mM KCl, 10 mM NaCl, 3 mM MgCl₂, 50 mM Tris acetate, pH 7.8, 70 mM glycine, 0.012% bovine lactalbumin, 10% glycerol) with 10 mM EDTA at 37°C for 0.5 or 24 h. Binding reactions were loaded onto 10% non-denaturing polyacrylamide gels and electrophoresis was performed at 70 V for 15 h in a cold room (7°C). Gels were imaged on a Typhoon 9210 Imager (GE Healthcare), and band densities were analyzed using Multi Gauge software (Fuji). The apparent K_d was derived from the Scatchard equation using SigmaPlot 11.0 software to perform direct curve- fitting analysis. For binding experiments in which the monovalent cation composition was varied, recombinant G4R1 at concentrations of 50–500 pM was incubated with 1 pM 5’-[32P]-end labeled G4-DNA and Res buffer with either 50 mM KCl/10 mM NaCl, 50 mM KCl or 50 mM NaCl. Analysis was performed as described above.