3.3. Biophysical Assays

Stabilization of compounds with quadruplex-structure was monitored via FRET-melting assay performed in 96-well plates on real time PCR apparatus 7900HT Fast Real-Time PCR System as follow: 5 min at 25 °C, then increase of 0.5 °C every minute until 95 °C. Each experimental condition was tested in duplicated in a volume of 25 µL for each sample. FRET-melting assay was performed with oligonucleotides that mimic the human telomeric sequence, as well as other quadruplex-forming oligonucleotides, equipped with FRET partners (here: FAM and TAMRA) at each extremity in the presence of the G4 ligand. To determine ligand G4 vs. duplex selectivity, an unlabeled DNA competitor ds26 was used. The oligonucleotides were prepared at 0.2 µM, the ligands at 1 µM, and competitors at 0, 3, and 10 µM final concentration. Measurements were made with excitation at 492 nm and detection at 516 nm in a buffer of lithium cacodylated (10 mM, pH 7.2), NaCl (100 mM) or KCl (10 mM, completed by 90 mM LiCl for F21T, and 1 mM, completed by 99 mM LiCl for all the others G-quadruplex sequences). To identify if the ligands are able to stabilize the G-quadruplex structure, we determined the temperature at half denaturation of the G4 in the absence and in the presence of ligand. The experiments were carried out on prefolded G-quadruplex structures: the sequences were heated at 90 °C for 5 min and left to cool down at 4 °C overnight. At last, to identify a preferential binding towards a particular G-quadruplex conformation, a series of labeled oligonucleotides covering a range of possible G4 conformations was used (Table 4):

DNA sequences employed for the FRET melting assay.

Cell G4-FID assays were performed in a fluorescence Agilent Cary Eclipse spectrophotometer. A temperature of 20 °C was kept constant with thermostated cell holders. Each experiment was performed in a 1 mL cell, in 10 mM lithium cacodylate buffer (pH 7.4) with 100 mM KCl or NaCl depending on the experiment, in a total volume of 1 mL. The G4-FID assay was designed as follows: 0.25 μM pre-folded DNA target was mixed with thiazole orange (0.50 μM for G4-DNA, 0.75 μM for ds26). Each ligand addition (from 0.5 to 10 equivalents) was followed by a 3 min equilibration time, after which the fluorescence spectrum was recorded. The percentage of displacement was calculated as follows: TO displacement (%) = 100 − [(FA/FA₀) × 100], where FA and FA₀ stand for the fluorescence emission area of TO bound to DNA after each ligand addition and FA₀ before ligand addition (area measured from 510 to 750 nm, λexc = 495 nm, slit = 10 nm). The percentage of displacement was then plotted as a function of the concentration of added ligand. As for the FRET melting assay a series of oligonucleotides covering a range of possible G4 conformations was used (Table 5).

DNA sequences employed for the G4-FID assay.

CD experiments were carried out at 20 °C with a JASCO J-710 spectropolarimeter equipped with a Peltier temperature controller (Jasco PTC-348WI) interfaced to a PC, by using 1 cm path rectangular quartz cells (1 mL reaction volume). Scans were recorded from 220 nm to 400 nm with the next parameters: 100 mdeg sensitivity, 1 nm data pitch, 200 nm min⁻¹ scan speed, 1 s response, 1 nm band width, and 4 accumulations. Solutions containing 5 μM 22AG were titrated with increasing concentration of ligands (0–5 eq, 10 min stabilization time) in lithium cacodylate (10 mM) supplemented with potassium or sodium chloride (100 mM) at pH 7.2. The scan of the buffer was subtracted from the average of the scan of the samples. The signal was further smoothened with a Savitzky–Golay method (2 order, 20-point window). The CD data were blank-subtracted and normalized to molar dichroic absorption (Δε) on the basis of concentration using Equation (1), with θ the ellipticity in millidegrees, c concentration in mol L⁻¹, and l the path length in cm [38]: