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, By cooling at room temperature, crystals separated that were filtered-off, washed successively with DMF (2 x 0.2 mL), ethanol (2 x 0.5 mL) and diethyl ether (2 x 2 mL) and dried
, standard analytical HPLC conditions) = 18.7 min
, CD spectra were recorded on a J-810 spectropolarimeter (Jasco) at 20°C, after of 500 nm.min -1 . They were baseline corrected by subtracting the spectrum of a water filled quartz cell and zero corrected at 340 nm. Titration experiments were performed by incremental additions of 3 µL of a 180 µM ligand solution, prepared in a 1X ad hoc buffer from a 5 mM stock solution in DMSO, in a 540 µL oligonucleotide solution. CD signal amplitudes were corrected for the dilution factor resulting from ligand additions. UV-melting profiles were recorded on an Uvikon XL spectrophotometer (Secomam) equipped with a circulating water bath (Julabo) and a dry airflow in the sample compartment. Samples were heated at 95°C for 2 min, cooled down at 5°C, and then heated back at 95°C, at a rate of 0.2°C.min -1 . UV absorbance was recorded at 245, 260, 273, 295 and 335 nm every 5 and pre-incubated 10 min at 4°C in a buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM DTT, 10% glycerol, 0.2 mg/mL BSA and 0.1 mM EDTA. Binding experiments were carried out in 10 µl samples prepared in a reaction buffer containing 50 mM HEPES, pH 7.9, 0.1 mg/mL BSA, and 100 mM NaCl and 2% glycerol. For the first set of experiments, radiolabeled oligonucleotides (2 nM) were first incubated for 5 min at room temperature (? 20°C) with various amounts of G4 ligands (0, 1.25, 2.5 or 5 µM final concentrations) before addition of RPA (100-200 nM final concentration), and further incubation for 20 min at room temperature, Oligonucleotide samples were prepared in a 10 mM cacodylic acid buffer at pH 7.2 in bi-distilled water (adjusted with LiOH), containing 100 mM NaCl or 10 mM KCl plus 90 mM LiCl, and placed in quartz cells (Hellma) with an optical pathway of 1 cm
Band intensities (I) were quantified using ImageQuant 5.2. For each G4 ligand concentration, the fraction (F) of radiolabeled oligonucleotide substrate bound to RPA was calculated as I oligo bound / (I oligo bound + I oligo free ) and the RPA binding inhibition or displacement as (F0-Fc)/Fc, where F0 is the fraction of oligonucleotide bound in the absence of the ligand and Fc the fraction bound in the presence of the ligand at the indicated concentration. Experiments were performed at least twice, with error bars representing the standard error on the mean, Gel was then dried and exposed to a storage phosphor screen that was scanned with Typhoon? FLA 95000 biomolecular imager (GE Healthcare), vol.34, pp.1942-1958, 2015. ,