Pct 818

The degradation profile of GROs upon addition of serum was analyzed at different times by PAGE and FRET analysis. For a highly sensitive detection on PAGE, GROs used in this experiment were labeled with 6-carboxyfluorescein (FAM) at 5′ end. Oligonucleotides (0.5 μg) were incubated for different times with 10 μL DMEM supplemented with 10% FBS. The reactions were stopped by adding 0.1 mM EDTA and incubating the mixture at 100 °C for 5 min. The reaction products were analyzed by 16% polyacrylamide gel electrophoresis (PAGE). Prior to loading the mixtures onto the gel, 3 μL of glycerol solution (30% v/v) was added. Gels were run at 100 V for 45 min in Tris-Glycine buffer (pH 8.3). Gels were imaged by the UV transilluminator of a ChemiDoc XRS System (Bio-Rad Laboratories) and analyzed with the QuantityONE software. As far as FRET experiments are concerned, FAM/TAMRA-labelled oligonucleotides (0.1 μM) were incubated in PBS at 37 °C in the presence of 10% FBS. Fluorescence spectra were recorded at different times of incubation on a FP-8300 spectrofluorometer (Jasco) equipped with a Peltier temperature controller accessory (Jasco PCT-818). A sealed quartz cuvette with a path length of 1 cm (2 mL) was used. Measurements were made with excitation at 492 nm and emission from 500 to 650 nm at 100 nm/min scan rate, with both excitation and emission slits set at 5 nm.

FID experiments were performed
at 20 °C
on a FP-8300 spectrofluorimeter (Jasco) equipped with a Peltier temperature
controller accessory (Jasco PCT-818). A sealed quartz cuvette with
a path length of 1 cm was used. The assay was designed as follows:
0.25 μM pre-folded DNA target was mixed with thiazole orange
(0.50 μM for G4s, 0.75 μM for double-stranded DNA).^{53 (link)} Each ligand addition (from 0.5 to 10 equiv)
was followed by a 3 min equilibration time, after which the fluorescence
spectrum was recorded. Measurements were made with excitation at 495
nm and emission from 510 to 650 nm, with both excitation and emission
slits set at 5 nm. The percentage of displacement was calculated as
follows: TO displacement (%) = 100 – [(F/F₀) × 100], where F stands
for the intensity of the fluorescence emission signal at 543 nm of
TO bound to the DNA after each ligand addition and F₀ without added ligand. The percentage of displacement
was then plotted as a function of the concentration of added ligand.
DC₅₀ values were designed as the required concentration
to displace 50% TO from each investigated DNA.

A solution containing 0.25 μM of prefolded RNA (TERRA G4) or DNA G4 target and 0.5 μM of TO in 20 mM KH₂PO₄ (pH 7.0) and 70 mM KCl was prepared in a 1 cm path-length cell, and the corresponding fluorescence spectrum was acquired in the absence and presence of increasing concentrations of BPBA (10 mM stock solution in pure DMSO). Each ligand addition (from 0.5 to 20 molar equiv) was followed by a 3 min equilibration time before spectrum acquisition. The FID experiment was extended to a duplex DNA model (Hrp₂₇), in this case, three equivalents of TO (0.75 μM) were added to an oligonucleotide solution (0.25 μM). Measurements were run at 20 °C on a FP-8300 spectrofluorometer (Jasco, Easton, MD, USA) equipped with a Peltier cell holder (Jasco PCT-818), using an excitation wavelength of 485 nm and recording the emission in the 500–650 nm wavelength range. Both excitation and emission slits were set at 5 nm. The percentage of TO displacement was calculated as TO displacement (%) = 100 − [(F/ F₀) × 100], where F₀ is the fluorescence in the absence of ligand and F the fluorescence after each ligand addition. The percentage of displacement was then plotted as a function of the ligand concentration, and DC₅₀ was calculated as the required concentration to displace 50% TO from each investigated DNA. Each titration was repeated at least in triplicate.