Y-27632 br were conducted after the interaction
were conducted after the interaction of 7ESTAC01 with the SL-DNA/GE and dsDNA/GE using DPV. The oxidation of 7ESTAC01 and simulta-neous determination of DNA damage was performed in acetate buffer at pH 4.2 using oxidation potentials from 0 to + 1.6 V; 0.05 V amplitude; 0.0167 s sample width; 0.5 s pulse period and 2 s quiet time. All the intercalation measurements of 7ESTAC01 and DNA/GE biosensors were done with 7ESTAC01 in solution. The reduction of 7ESTAC01 was performed in acetate buffer at a potential range from 0.0 to − 0.122 V for 100 s before the oxidation process.
2.6. Interaction of ctDNA with 7ESTAC01 by UV–Vis spectroscopic and molecular docking studies
UV–Vis Y-27632 spectroscopy was performed at a fixed 20 μM 7ESTAC01 while varying ctDNA from 0 to 20 μM. The concentration of the stock solution of ctDNA (0.38 mM per nucleotide) was determined by UV absorption, using a molar extinction coefficient of 6600 M−1 cm−1 at 260 nm. A ratio > 1.8 at A260/A280 was obtained as indicative that DNA was sufficiently free of proteins. The intrinsic binding con-
stant (Kb) of the compounds with ctDNA was calculated according to
where [DNA] is the concentration of DNA per nucleotides, a is the molar absorption coefficient of the complex at a given DNA con-centration (Aobs. / [Compound]), f is the molar absorption coefficient of the complex in free solution, and b is the molar absorption coeffi-cient of the complex when fully bound to DNA. A plot of equation (Eq.
(2)) allows the determination of the intrinsic binding constant Kb, ob-tained by the linear data fit. The value of constant was calculated as the ratio between the slope and the intercept.
All molecular dynamic and Density Function Theory (DFT) calcu-lations were performed in agreement with Silva et al., (2016, 2017). The coordinates for building the molecular model were extracted from the X-ray crystal structure of the ctDNA dodecamer d(CGCGAATTC GCG) (PDB entry: 1BNA). Gold v.5.4 software from Cambridge Crystallographic Data Centre (CCDC) was utilized to perform all mo-lecular docking studies (Huang et al., 2013). Initially, all hydrogens were added into the DNA structure and, then, 7ESTAC01 (ligand) was introduced into space. Different genetic algorithms (GA) were applied to find the best score function for the ligand. The GoldScore, Chem-Score, Piecewise Linear Potential (ChemPLP), and Astex Statistical Potential (ASP) functions were employed to obtain the best 10 binding
After the docking calculations, the DFT calculations were performed using quantum mechanics (QM) models from the Spartan'14 program to determine the corrected free binding energy (ΔG) for the ctDNA-7ESTAC01 complex. The potential of intercalation of 7ESTAC01 was investigated by theoretical methods. The optimized geometries of this compound's ability to interact with the ctDNA were taken from docking analysis. In addition, the coordinates of the ctDNA structure were taken from the crystal structure (PDB ID: 1BNA), and the ligand and water molecules were removed. The binding energy of the DNA/ligand complex was calculated by applying the M06/6-31G (d) basis set. The M06 method employed the global hybrid functional, which is the top performer within the 6 functionals of the main group, thermochemistry, kinetics and non-covalent interactions. Moreover, frequency calcula-tions were performed to confirm the nature of the stationary point at the same level. QM binding energies were obtained applying the
following formula, in which the free binding energy of Gibbs (ΔG) was calculated as the difference between the energy of the complex (EDNA-
ligands) and the sum of the ctDNA (EctDNA) and ligand (Eligand) energies based on the equation (Eq. (3)). G = [EDNA
The final energy of the optimized structure was improved by in-cluding the single point energy from the 6-31G(d) basis set unscaled zero-point energy (ZPE) and thermal corrections (at 298.15 K and 1 atm) estimated at the same level of theory, using the Spartan'14 program. All these protocols were performed exactly as described by Silva-Júnior et al. (2017).
3. Results and discussion
3.1. Electrochemical characterization of 7ESTAC01
The electrochemical characterization of acridine-9-carboxaldehyde with 2-aminothiophene derivative designed as 7ESTAC01 (Fig. 1A) was investigated using CV on the GE in a mixture of phosphate buffer (pH 7.2) and 20% DMF in nitrogen saturated solutions. CVs were registered in the range from − 1.8 V to + 1.0 V (Fig. 1B). Fig. 1B shows the re-duction of 7ESTAC01 displaying two waves. The first one exhibits a quasi-reversible reduction peak at EIc = −0.38 V vs. Ag/AgCl with a peak separation potential (ΔE = Ea-Ec) of 150 mV. The second one displays an irreversible reduction peak at EIIc = −0.71 V. In the same way, 9-aminoacridine, which was synthesized containing only acridine, was examined in the same potential range from − 1.8 V to + 1.0 V (Fig. 1B, inner graph). The first cathodic potential for the 9-aminoa-cridine showed a single peak at EIc= −1.2 V, Fig. 1B (inner graph). It is important to mention that no wave was registered in the oxidation potential range for the 9-aminoacridine. Thus, only the reduction po-tential range was further studied.