br For CD melting procedures AT
For CD melting procedures, AT11-B0 solutions were prepared in 10 mM KCl and 90 mM LiCl in order to obtain a melting temperature around 50 °C. The denaturation process was analyzed by monitoring the wavelength of maximum ellipticity (260 nm) through temperatures ranging from 10–20 to 110 °C (temperature set by the Peltier system) with a heating rate of 2 °C/min, in the presence or absence of acridine orange derivatives.
Data were converted into fraction folded (θ) plots according to Eq. (1), fitted to a Boltzmann distribution using OriginPro2016:
The melting temperatures (Tm) were determined from a two-state transition model where CD is the ellipticity at 260 nm at each tem-perature and CDmin and CDmax are the lowest and highest ellipticities, respectively.
2.4. Fluorescence binding studies
Fluorescence titrations were performed on a Horiba FluoroMax4 fluorometer (Japan) using a high-precision quartz suprasil cuvette (light path 10 mm × 4 mm). For these experiments, previously an-nealed AT11 or AT11-B0 at concentration of 500 µM in 20 mM po-tassium phosphate buﬀer containing 65 mM KCl were successively added to a solution of the same buﬀer containing 5 µM of one of the acridine orange ligands. After each addition the mixture was left for 10 min for equilibration. All spectra were acquired with an Voriconazole time of 0.5 s, an emission and excitation slit fixed at 2 nm and step size of 1 nm, averaged over three scans. The acridine orange ligands were excited at 498 nm and the emission spectra were collected from 530 to 700 nm. Additionally, Cy5-labelled AT11 and AT11-B0 at 1 μM were titrated with nucleolin in the absence and presence of C8 to evaluate the eﬀect of ligand binding in the aﬃnity of the G4 structures towards the protein. The fluorescence emission spectra were obtained between 660 and 800 nm and averaged over three scans.
The obtained fluorescence data was converted into fraction of li-gand bound (α) plots according to Eq. (2):
I − Iλfree
where I is the fluorescence intensity at 568 nm at each ligand:DNA
J. Figueiredo, et al.
ratio, and Ifree and Ibound are the fluorescence intensity of the free and fully bound ligand, respectively. Data points were then fitted to Hill saturation binding model (OriginPro 8):
where KD is the apparent equilibrium dissociation constant, [DNA] is the concentration of the DNA and n is the Hill constant which describes cooperativity of ligand binding. For the protein binding experiments, data points were fitted to Michaelis-Menten model (OriginPro 8):
2.5. Serum stability assay
2.6. Cell viability assay
Normal human dermal fibroblasts (NHDF) were grown in RPMI medium, supplemented with 10% FBS, 1% streptomycin-penicillin an-tibiotic, 0.01 M HEPES, 0.02 M L-glutamine and 0.001 M sodium pyr-uvate. Additionally, cervical cancer cell line (HeLa) was grown in DMEM medium, supplemented with 10% FBS and 1% streptomycin-penicillin antibiotic. Cell cultures were maintained in a controlled hu-midified atmosphere at 37 °C and 5% CO2. Cells were plated in 48-well culture plates (HeLa or NHDF were seeded at a density of 0.5 × 104 cells/mL or 1 × 104cells/mL, respectively) in 300 µL of medium. In the next day, both cell lines were treated with the pre-formed AT11 or AT11-B0-ligand complexes (2 µM of C3/C5 or 1 µM of C8 with 15 µM of AT11 or AT11-B0 for 10 min) and incubated for 7 days. At the end of incubation, 50 μL of thiazolyl blue tetrazolium bromide (MTT) solution was added to each well and further incubated for 1 h in HeLa or 4 h in NHDF cells. The resulting formazan crystals were solubilized with 250 μL of DMSO and the optical density (OD) was measured at 570 nm. Cell viability was normalized to control condition (cells without li-gands). Statistical analysis was performed by using Student’s unpaired t test. The values of p < 0.05 were considered statistically significant. Data analysis was performed with GraphPad Prism 6 software (San Diego, CA, USA).
2.7. Confocal fluorescence microscopy
To better understand the internalization ability of free AT11 or AT11-B0 and complexes, HeLa and NHDF cells were seeded at a density of 0.3 × 104 and 5 × 104 cells/mL, respectively, in treated 8 well µ-slides (IBIDI, Germany). Cells were grown at 37 °C in 5% CO2 humi-dified atmosphere and treated the day after with the pre-formed com-plex (0.5 µM of acridine orange ligands (C3, C5 or C8) and 1 µM of AT11 and AT11-B0), ligand-only control (0.5 µM) and DNA-only (AT11 and AT11-B0) controls (1 µM). After 3 days of incubation, cells were washed with phosphate-buﬀered saline and nuclei were then stained with 1 µM Hoechst 33,342 nuclear probe for 15 min. Cells were then observed using a Zeiss AxioObserver LSM 710 microscope.