Fluorescent Titration Shift Assay Protocol

FTS experiments were based on the method described by Müller et al^{16 (link)}. Gels were prepared with 50 mM Tris-acetate (pH 7.5), 10 mM MgAc₂, and 10% (19:1) acrylamide/bis-acrylamide, then polymerized as described above. RNAs and tet/dox were combined and diluted to assay concentrations in binding buffer (20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl₂), denatured at 65°C for 5 min, and cooled at room temperature for 45 minutes. Following incubations, 16.7% gycerol was added and vortexed to weight samples for gel loading. Gels were pre-run for 30 min at 1W, loaded, and samples separated for 6h at 1W. Following electrophoresis, gels were transferred to a ChemiDoc XRS gel imaging system (Bio-Rad #1708265). Tet/dox fluorescence was imaged using UV excitation and a 530/28 nm filter. Gels were then stained for 5 min using 0.2X SYBR-Gold (Thermo #S11494), washed, destained for 10 min, and imaged using SYBR-Gold filter settings. Composite gel images were generated using ImageJ.