Rx2000 stopped flow apparatus

All steady-state fluorescence spectroscopic experiments were measured on a Cary Eclipse spectrometer (Varian, Australia) equipped with a peltier block, a magnetic stirring device and a RX2000 stopped-flow apparatus (Applied Photophysics Ltd, UK). The data obtained were processed with OriginPro 2018 software (OriginLab, USA). Aminopurine-modified RNA samples were prepared in 0.5 μM concentration in a total volume of 120 μl (qualitative analysis) or 1 ml (quantitative analysis) of buffer (50 mM MOPS, 100 mM KCl, pH 7.5 (25°C)). The samples were heated to 90°C for 2 min, allowed to cool to room temperature, transferred to quartz cuvettes equipped with a small stir bar and held at 20°C in the Peltier controlled sample holder. Then, ligands were manually pipetted in a way not to exceed a total volume increase of 3%. For binding affinity experiments, the solution was stirred after ligand addition and allowed to equilibrate for at least 15 min before data collection. Spectra were recorded from 320 to 500 nm using the following instrumental parameters: excitation wavelength, 308 nm; increments, 1 nm; scan rate, 120 nm min⁻¹; slit widths, 10 nm. Thermodynamic and kinetic parameters K_d and k_obs were obtained as described in reference (20 (link)).