Binding and thermodynamics of REV peptide−ctDNA interaction

2.1 Biochemicals

Highly polymerized fibrous ctDNA was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). A stock solution was prepared by dissolving ctDNA in 2–5 mL of sterile autoclaved water by mixing overnight at room temperature. The concentration of ctDNA was determined spectrophotometrically by measuring absorbance at 260 nm, 25 °C using molar extinction coefficient ε₂₆₀ = 13,200 bp cm⁻¹ M⁻¹ (base pair).21 Absorption spectra were recorded on Agilent Cary 100 Series UV–Vis Spectrophotometer. The purity of ctDNA was determined by the ratio of absorbance at 260 nm to that at 280 nm. The observed A260/A280 >1.8 for ctDNA solution indicates a protein-free solution.22, 23 Protein-free purity of ctDNA was also confirmed using gel electrophoresis based analysis on a 10–15% SDS-PAGE stained with Coomassie Brilliant Blue (data not shown).

REV peptide (DTRQARRNRRRRWRERQRAAAR) was purchased from Beijing SBS Genetech Co. Ltd., Beijing, China and used as obtained. REV peptide (22 mer) was synthesized by solid-phase peptide synthesis using Fmoc-Chemistry and purified by HPLC using 0.1% trifluoroacetic acid in 100% water and acetonitrile, with 1.0 mL min⁻¹ flow rate, at 214 nm wavelength, and with a 100 μL load volume. The REV peptide was obtained in a lyophilized powdered form with a purity of 95.06%. All stock solutions were diluted to the required concentrations with 10 mM sodium phosphate buffer, pH 7.0. All chemicals were of analytical reagent grade and autoclaved ultrapure water was used throughout the study.

2.2 Circular dichroism (CD) spectroscopy

To detect conformational changes observed between ctDNA and REV peptide, CD spectral data were collected using JASCO (model J815) spectropolarimeter equipped with a computer-controlled thermoelectrical cell holder using 1 cm path length quartz cuvette (Hellma GmbH & Co. KG, Müllheim, Germany). CD spectra of ctDNA in the absence and presence of varied concentrations of REV peptide were recorded at wavelengths ranging from 350 nm to 200 nm under constant nitrogen flush at 25 °C. A scan speed of 500 nm min⁻¹, 1 nm bandwidth, 1 nm data pitch, and 3 accumulations with a response time of 1 s were adopted. Samples were prepared in 10 mM sodium phosphate buffer, pH 7.0. The concentration of ctDNA was fixed at 50 μM while REV peptide concentration was varied from the charge ratios (Z _+/−) of 0.05–3.6. In a quartz glass CD cuvette, 500 μL buffer (10 mM sodium phosphate buffer, pH 7.0) was added and CD spectra were recorded for the baseline. A solution of 50 μM ctDNA was added to CD cuvette and mounted on the CD spectrometer to record CD spectra of ctDNA. REV peptide was added in different charge ratios (Z _+/−) to same CD cuvette containing ctDNA solution and the whole solution was mixed well by inverting the cuvette up and down several times. The CD spectra measurement for the complex was repeated with 5 min incubation interval for each of the charge ratios (Z _+/−).

2.3 Temperature-dependent UV spectroscopy (UV melting studies)

All melting measurements were carried out using Agilent Cary 100 Series UV–Vis Spectrophotometer with a temperature rate of change at 0.5 °C min⁻¹. The instrument reading was zeroed with no reference. ctDNA–REV peptide complex melting was followed by changes in UV absorbance. A quartz cuvette (Hellma GmbH & Co. KG, Müllheim, Germany) of 1 cm path length was used for all melting studies. The ctDNA concentration was kept constant at 25 μM while REV peptide concentration was varied from molar ratios 0.5 to 3.5 with respect to ctDNA. Temperature-dependent absorption spectra were recorded at 260 nm at the rate of 0.5 °C min⁻¹ increase in temperature from 40 °C to 95 °C in 10 mM sodium phosphate buffer, pH 7.0. The melting temperature (T_m) for each optically detected transition was calculated using the previously described method.24

2.4 Steady-state fluorescence spectroscopy

Fluorescence spectra were measured on Horiba Fluoromax-4 spectrofluorometer equipped with thermoelectrically controlled cell holders at a scanning speed of 80 nm s⁻¹. The excitation wavelength of the sample was 480 nm and the fluorescence emission spectra were collected in the range 500 nm–700 nm. A quartz cuvette (Hellma GmbH & Co. KG, Müllheim, Germany) of 1 cm path length, transparent from all four sides, was used throughout. All experiments were carried out at 25 °C. ctDNA concentration was fixed at 50 μM and the REV peptide concentration was varied from the charge ratios (Z _+/−) of 0.05–3.6 μM in 10 mM sodium phosphate buffer, pH 7.0. 50 μM ethidium bromide (EtBr) was used as a fluorescent probe.25 All solutions were mixed and incubated for 5 min before measurements were recorded.

2.5 Isothermal titration calorimetry (ITC)

ITC experiments were carried out on VP-ITC Micro Calorimeter (MicroCal, Northampton MA) at different temperatures 293 K, 298 K, 303 K, and 308 K. The interaction between ctDNA and REV peptide was also investigated at different Na⁺ concentrations (0.016 M, 0.026 M, 0.056 M, and 0.106 M) at 298 K in 10 mM sodium phosphate buffer, pH 7.0. Sodium chloride was used to perform variable salt-dependent ITC experiments. Before loading, the solutions were thoroughly degassed. The titration cell was filled with a solution of 0.1 mM ctDNA in 10 mM sodium phosphate buffer, pH 7.0. The syringe was loaded with 3.0 mM REV peptide solution in the same buffer. Titrations of ctDNA with REV peptide were carried out by injecting 10 μL aliquots of 3.0 mM REV peptide from rotating syringe at a speed of 307 rpm. Total 25 syringe injections containing REV peptide were titrated into the cell containing ctDNA at 180 s time interval between repeated injections. The ITC equilibrium options were set for fast and auto mode. ITC data was analyzed with Origin 7.0 and the binding parameters were determined. The one-site binding model was used to fit the integrated data to obtain the stoichiometry and binding constants. The binding affinity (K_b) values were obtained and the corresponding ΔG values were calculated from the equation:

(1)

Where ΔG is the change in standard Gibbs free energy, R is the universal gas constant (R = 1.987 cal mol⁻¹ K⁻¹), T is the temperature (in Kelvin) and K_b is the binding constant.

3.1 Secondary structure rearrangement by REV peptide

We adopted CD spectroscopic analysis to reveal structural and conformational changes of ctDNA upon REV peptide ligand binding. The CD spectra of ctDNA were collected in the presence of REV peptide at different concentrations of REV peptide (Figure 1). The molar ellipticity of native ctDNA shows four major bands at 270 nm (positive), 245 nm (negative), 220 nm (positive), and 210 nm (negative). The positive CD signal at 270 nm accentuates, but the positive signal at 220 nm inverts to a negative value during formation of ctDNA–REV peptide complex. The negative signal at 210 nm accentuates further during complex formation. However, the signal at 245 nm remains nearly unchanged.

The observed prominence in the new intensity of negative peak at 220 nm and in positive peak at 270 nm, suggests a decrease in base stacking interactions.26 Interestingly, the CD spectrum undergoes a sharp change and shifts from 270 nm to 285 nm and the peak area increases at a charge ratio (Z_+/−) greater than 1.3. This change in molar ellipticity suggests large conformational changes of ctDNA molecule on binding between ctDNA and REV peptide. The changes in ctDNA–REV peptide complex take place in a concentration-dependent manner. The molar ellipticity at wavelength 270 nm versus charge ratios (Z_+/−) plot is shown in Figure 2. Two binding modes are evident. At the charge ratio (Z_+/−) of 1.0, the ctDNA:REV peptide exhibits 1:1 stoichiometry (Mode I), whereas the complex evidently has 1:3 stoichiometry (Mode II) at the charge ratio (Z_+/−) of 3.0. No further conformational changes were observed at charge ratios >3.0 suggesting the reach of saturation point of the complex formation.

3.2 ctDNA thermal denaturation in the presence of REV peptide

We utilized UV–Vis absorption spectroscopy to understand the stability of ctDNA binding with REV peptide. The goal was to probe the thermodynamic stability of secondary structure in the ctDNA–REV peptide complex. Thermal denaturation of ctDNA was carried out in the presence of different concentrations of REV peptide to observe effects of binding on the stability of the structure. The melting profiles at 260 nm of ctDNA and its complex with the REV peptide at different concentrations are shown in Figure 3. The absorbance at 260 nm increases with the rise in temperature. The melting curve was sigmoidal and showed a shift indicating hyperchromic effect at 260 nm. The absorbance profile of ctDNA–REV peptide complex shows saturation at higher temperature. The melting temperature (T_m) of the native ctDNA was obtained at 75.5 °C. Upon binding to REV peptide, the melting temperature of ctDNA increased proportionally to the REV peptide concentration. The changes were observed in melting temperatures of ctDNA from 75.5 °C to 77 °C, 77.5 °C, 83 °C, and 83.5 °C for ctDNA:REV peptide complex at molar ratios of 1:0, 1:0.5, 1:1, 1:1.5, and 1:3.0, respectively. No significant differences were observed for ctDNA:REV peptide molar ratios above 1:3.0 (Figure 3). As depicted in Figure 3, a similar trend was observed for ctDNA:REV peptide molar ratios 1:3.0 and 1:3.5. This is indicative of saturation phase of the complex formation as observed with CD and fluorescence results (see section 3.3) in which further complex formation was not observed after Mode II. The significant change observed in melting temperature of ctDNA on the addition of REV peptide shows higher stabilization of the ctDNA–REV peptide complex.

3.3 Fluorescence binding studies

To gain additional insights into the binding between ctDNA and REV peptide, steady-state fluorescence spectroscopy was employed. The binding was characterized using EtBr as a fluorescence spectroscopic probe. EtBr acts as an intercalating agent and was used as a probe to identify the displacement of bound EtBr–ctDNA complex fluorescence intensity by REV peptide.25 All fluorescence titration studies were carried out in 10 mM sodium phosphate buffer, pH 7.0 at 25 °C. The fluorescence titration was performed keeping ctDNA concentration constant and the REV peptide was added in increasing concentrations. Fluorescence titration spectra indicating EtBr displacement from EtBr–ctDNA complex with the successive addition of REV peptide are shown in Figure 4. Increasing the concentration of REV peptide results in gradual decrease in the fluorescence intensity of EtBr-bound ctDNA due to the displacement of fluorescence intensity of complex, suggesting quenching.27 The changes in ctDNA fluorescence intensity of the EtBr-bound ctDNA at 610 nm was normalized and variation of I₀/I plotted against charge ratios (Z_+/−), where the quantity of complex formed increased until all the available ligand was bound (Figure 5). As depicted in Figure 5, it is evident that, similar to CD results, two types of binding modes were observed in steady-state fluorescence studies. The 1:1 (Mode I) and 1:3 (Mode II) stoichiometries of ctDNA:REV peptide complex were observed at the total REV peptide molar ratios of 1.0 and 3.0, respectively. The fluorescence intensity of EtBr-bound ctDNA decreased upon addition of REV peptide and saturation was observed beyond REV peptide molar ratio (Z_+/−) of 3.0 (Figure 5). The fluorescence measurements suggest that binding between ctDNA and REV peptide occurs maximum up to REV peptide molar charge ratios (Z_+/−) of 3.0, and beyond this ratio, REV peptide does not bind ctDNA any further.

3.4 Equilibrium binding studies by isothermal titration calorimetry (ITC)

To understand the thermodynamic basis of ctDNA binding with REV peptide, isothermal titration calorimetry (ITC) studies were performed. Figure 6 shows the results of a typical titration curve obtained at 298 K. The heat burst generated after addition of 3.0 mM REV peptide to the solution containing 0.1 mM ctDNA at temperature 298 K and the temperature variable (293 K–308 K) of ITC profile is depicted in Supporting Information, Figure S1. The major thermodynamic parameters related to ctDNA–REV peptide binding are presented in Table 1. The best fitting data for ctDNA binding isotherm with a model for one site is shown in Figure 6 and Supporting Information, Figure S1. The ctDNA–REV peptide binding affinity (K_b) obtained during all ITC experiments was positive value (Table 1), indicating specific binding. The change in enthalpy (ΔH) was negative value, indicating that ctDNA–REV peptide binding is a heat releasing exothermic process. The value of change in Gibbs free energy of binding (ΔG) was calculated using Eq. 1 (see Materials and Methods section 2.5) and the value is presented in Table 1. TΔS upon binding was calculated using the thermodynamic standard Gibb's equation ΔG = ΔH – TΔS. The entropy change (TΔS) associated with the binding between ctDNA and REV peptide was small positive values depicted in Table 1.

The binding affinity (K_b) measured from ITC studies was 3.9 (±0.02) × 10⁶ M⁻¹ at 298 K. At all the temperatures (293 K–308 K), and variable salt ITC experiments, the binding enthalpies (ΔH) were found to be negative, with nearly equal values. However, in all cases, the binding entropy (ΔS) was found to be favorable positive value. The stoichiometry of binding was calculated from the fitting of binding isotherm which gave a value of n = 3.3, suggesting 1:3 stoichiometry of ctDNA:REV peptide complex. Nearly similar results were obtained in most of the temperatures and salt variable ITC experiments. The 1:3 stoichiometry of the complex suggests that 3.0 REV peptide molar charge neutralizes one molar charge of ctDNA.

3.5 Heat capacity (ΔC_p) measurements

The temperature dependence of the enthalpy was studied by ITC at different temperatures to determine the molar heat capacity (ΔC_p). The ITC curve and heat burst generated at different temperatures ranging from 293 K to 308 K are shown in Supporting Information, Figure S1. The relationship between enthalpy and temperature is depicted in Figure 7A and Supporting Information, Figure S2. The ΔC_p value during the binding interaction between ctDNA and REV peptide was determined from the temperature dependence of the observed binding enthalpy using the standard relationship:

(2)

The solid line denotes the linear regression fit of the data points. The slope of the resulting line of the plot between enthalpy change (ΔH) vs temperature (K) is depicted in Supporting Information, Figure S2 shows ΔC_p value of 0.007 kcal mol⁻¹ K⁻¹ for the binding of ctDNA with REV peptide. This value suggests negligible changes in heat capacity, and therefore, ΔC_p is considered to be close to zero. The value obtained for ΔC_p is an exceptional case for ITC-based heat capacity experiments. In addition, the effect of temperature on the entropy (ΔS), enthalpy (ΔH), and change in Gibbs free energy (ΔG) components are given in Figure 7A. The TΔS component was observed to be independent of ΔH and ΔG (Figure 7B). A pictorial representation of the standard thermodynamic relationship ΔG = ΔH − TΔS (Figure 7B) shows that REV peptide binding to ctDNA is favored by entropy.

3.6 Salt dependence of binding constants

Furthermore, ctDNA–REV peptide binding studies were carried out at different Na⁺ concentrations using ITC (see Materials and Methods section 2.5) at 298 K as presented in Supporting Information, Figure S3. The data derived from the ITC experiment performed at the highest salt concentration (0.106 M) is shown in Figure 8. A minor decrease in the thermodynamic parameters such as K_b, ΔH, TΔS, and ΔG_obs was observed. The ctDNA–REV peptide interaction at any given salt concentration has a favorable entropic and enthalpic contribution to the observed free energy. Similar to variable temperature-dependent experiment, the enthalpy change was found to be negligible during variable salt-dependent ITC experiment. The thermodynamic data observed at 298 K during variable salt ITC experiment is summarized in Table 2. The observed binding constant (K_b) decreased upon increasing salt [Na⁺] concentration from 0.016 M to 0.106 M. The enthalpic and entropic contributions to the free energy in the range of salt concentrations used are shown in Table 2. The observed free energy decreases while increasing the salt concentration, whereas the enthalpy remains almost constant in each case indicating that decrease in binding affinity is an entropically driven phenomenon.

3.7 ctDNA–REV peptide binding is governed by electrostatic interactions

Thermodynamic studies of molecular interactions yields valuable information about the type of interactions that govern binding events. One of the important interactions observed in DNA ligand binding is electrostatic, which arises due to electric force between two charged objects. To gain insight into the electrostatic interaction and involvement of cationic charges, ITC was further used to understand the effect of salt (NaCl) upon ctDNA–REV peptide binding. The plot of lnK_b versus ln[Na⁺] in Figure 9A clearly shows a decrease in K_b with increasing salt concentration. This decrease in K_b is due to counter ion release during the interaction of charged ligand.28 The polyelectrolyte theory28 can explain this behavior of interaction in which the interacted free energy (ΔG_obs) dissects into its two components—electrostatic (ΔG_pe) and nonpolyelectrolyte (ΔG_t). The correlation of interacted free energy of interaction with its two components can be expressed as:

(3)

The slope of the lnK_b versus ln[Na⁺] plot (Figure 9A) is given by:

(4)

In Eq. (4), SK is the slope, Z is the charge on the ligand involved in the interaction, and Ψ is the proportion of counterions for the first fit associated with each phosphate group of DNA molecule. The counterion on the first fit Ψ value for double stranded B-type DNA is normally 0.88.

The predicted SK for the plot in Figure 9A is −3.53. Figure 9A indicates the charge value (Z) = +4.01 for REV peptide at neutral pH.

The electrostatic or polyelectrolyte free energy term is given by the equation 28:

(5)

All the thermodynamic parameters obtained during variable salt measurements are depicted in Table 2. The electrostatic free energy component (ΔG_pe) was calculated using Eq. (5) as:

(6)

where [Na⁺] was 0.056 M. The nonpolyelectrolyte contribution (ΔG_t) was then calculated using Eq. (3).

The calculated ΔG_pe value was −6.0 ± 0.6 kcal mol⁻¹. On analyzing thermodynamic parameters which contributed to ΔG_obs, it is clear that nearly 73% of the observed binding free energy results from electrostatic interactions while nearly 27% of it originates from nonpolyelectrolyte interaction. The data presented in Figure 9B confirms that the interaction is of electrostatic type as ΔG_pe > ΔG_t. Also, the slope (Figure 9A) and the intercept of linear least-squares regressions suggest that the binding interaction is typically electrostatic in nature. The charge value (Z) = +4.01 confirms that only 3–4 Arg out of 11 charged Arg from REV peptide participate during interaction with ctDNA. It seems that REV peptide binds electrostatically to the phosphate of ctDNA and binding happens with the bases present in groove region of ctDNA.

4.1 REV peptide induces the Ψ conformation of ctDNA

The DNA–ligand interactions are important to understand the effect of ligand on replication and transcription process, which in particular are of interest for the rational design of anticancer and antibacterial drugs. Most chemotherapeutic pharmaceuticals are derived from DNA binding ligands which interact with the DNA duplex by different modes of binding namely DNA intercalation, groove binding, covalent binding, and electrostatic interaction. Therefore, CD spectroscopy is extremely useful in performing binding and conformational studies of nucleic acids and for specific ligands that recognize DNA sequences and structures with high selectivity.30-34

Two CD signals of ctDNA observed at 220 and 270 nm are accentuated during interaction with REV peptide. The positive band at 270 nm is due to base stacking and the negative band at 245 nm is due to the helicity of the right-handed B form of ctDNA structure.35 These results show significant changes in base stacking and helicity during interaction with REV peptide. The alteration in CD spectral band position as well as intensity is due to the large conformational transitions observed in duplex ctDNA during its binding with REV peptide. The CD spectral positive band at 220 nm and another positive band at 270 nm experienced change in orientation and position suggesting significant distortion in native conformation of B form structure of ctDNA due to its interaction with REV peptide,34, 35 even at a low charge ratio. When the charge ratio was increased to >1.3, the CD spectrum experienced a shift from 270 nm to 285 nm, indicating a characteristic morphological change in ctDNA from B to Ψ form.34 This change observed in the presence of REV peptide is likely due to DNA condensation by REV peptide. This change could occur due to several factors such as concentration, preparation method, source, length, and pH of the solutions used for the study. The acidic pH or neutral pH favors B to Ψ transition. Another possibility is that the binding between ctDNA and REV peptide is a multiple event.

The plot of molar ellipticity change in CD spectrum at 270 nm against overall charge ratio (Z_+/−) provides us with valuable information about different binding modes whereby molar ellipticity change saturates at a charge ratio ≥3. Overall, two binding modes can be proposed based on CD spectral data for ctDNA–REV peptide complex, namely 1:1 (Mode I) and 1:3 (Mode II). This conclusion is somewhat similar to multiple binding stoichiometries and affinities reported earlier for ctDNA.36, 37 Three molar charge of REV peptide ligand is saturation limit per molar charge of ctDNA molecule. At the saturation limit of 1:3 of ctDNA–REV peptide complex, the overall structural morphology of ctDNA transits from B to Ψ conformation.

4.2 REV peptide stabilizes ctDNA

Temperature-based denaturation of DNA could be due to either electrostatic repulsion of two strands in the helix or overall gain in entropy during the change from double stranded DNA helix to single stranded random coil structure. Thermal melting studies support the stabilization of ctDNA structure upon binding with REV peptide. The complex with ctDNA–REV peptide molar ratio from 1:0.5, 1:1, and 1:1.5 experienced a T_m shift of 1.5 °C, 2.0 °C, and 7.5 °C, respectively. At ctDNA–REV peptide molar ratio of 1:3.0, the complex exhibited high thermal stability (T_m shift 8 °C). At a higher ctDNA:REV peptide molar ratio of 1:3 and above, a similar trend of melting profile was observed which indicates saturation phase of the complex. The higher stability of ctDNA–REV peptide complex is due to the formation of extra H-bonds and other noncovalent interactions between ctDNA and REV peptide. In addition, it is possible that polarity of REV peptide and ctDNA in this medium could have influenced the ctDNA melting. The dielectric constant difference of the medium with the rise in temperature could influence the binding forces and the energy of ctDNA-REV peptide binding interaction.

4.3 ctDNA–REV peptide saturation upon binding

The EtBr bound form of ctDNA upon complexation with REV peptide showed decrease in fluorescence intensity due to quenching,27 and saturated with REV peptide charge ratio Z_+/− = 3.0 or above (Figure 5). The shifting of midpoint of the fluorescence binding toward the higher concentration of REV peptide confirms the tight binding of the complex. The plot of variation of I₀/I as a function of EtBr–ctDNA complex concentration versus total REV peptide charge ratios (Z_+/−) provides insights on existence of two binding modes similar to an earlier report.35, 36 The complex achieves saturation of fluorescence at three molar charge of REV peptide to one molar charge of the ctDNA. Similar conclusions were apparent from CD spectroscopic data. The different modes of binding for ctDNA–REV peptide complex suggest multiple binding events. The saturation phase of the complex at charge ratio Z_+/− 3.0 or above suggests completion of charge neutralization. This is due to the hydrophobic interactions between positively charged REV peptide and ctDNA base pairs. It can be hypothesized that at higher charge ratios above 3.0, when charge neutralization process was complete, the REV peptide helped to condense ctDNA due to crowding effect around DNA chains. This results in the displacement of major portion of the intercalated EtBr molecule complexed with ctDNA helix.

4.4 Thermodynamics of ctDNA–REV peptide binding

The thermodynamics of binding between DNA and charged peptide can provide valuable information to understand biological processes. The thermodynamics of ctDNA and REV peptide binding can be considered as two continuous processes in a single ITC experiment. First, the cationic ligand, REV peptide, binds to the negatively charged ctDNA and then after reaching the critical ligand concentration, ctDNA starts condensation process. The charged ligands play a significant role while interacting with DNA molecule sensitive to electrostatic effects. Positively charged cationic peptides are condensed around the polyanionic DNA double helix in such a way that they form a mobile charge cloud around the DNA backbone and thus neutralize the charge. Therefore, DNA and cationic ligand binding are considered to be thermodynamically linked effects.

The polyelectrolyte theory and its effect on binding constants are depicted in Figure 9, which clarifies the contribution of polyelectrolytic (electrostatic) and nonpolyelectrostatic interactions.28 The ctDNA–REV peptide interaction is electrostatic as its contribution to free energy is more than nonpolyelectrostatic (ΔG_pe > ΔG_t). The experimental data suggests that out of −8.2 kcal mol⁻¹ free energy, −6.0 kcal mol⁻¹ and −2.2 kcal mol⁻¹ is due to electrostatic and nonpolyelectrostatic effect on ctDNA–REV peptide solution with 0.056 M NaCl solution, respectively. This could be due to release of condensed counter ions from the ctDNA helix upon REV peptide binding. The less contribution of nonpolyelectrostatic effect to ΔG_obs could be explained due to contribution of hydrogen bonding, van der Waals, and hydrophobic interactions. The presence of salt in the buffer is another factor for electrostatic contribution to the entropy changes in ctDNA–REV peptide interaction. The stability of ctDNA–REV peptide is due to the network of hydrogen bond and minor groove contacts achieved through van der Waals and hydrophobic interactions. Out of 11 Arg cationic counterion, only 3–4 Arg participate in the interaction due to charge value (Z) = +4.01 obtained for REV peptide at neutral pH (Figure 9A).

The sigmoidal binding isotherms obtained from ITC experiments are indicative of a single binding site. In all individual ITC experiments that were performed, the ctDNA:REV peptide stoichiometry was found to be 1:3 confirming the participation of 3.0 molar REV peptide charge per molar charge of ctDNA. Surprisingly, in all temperature variable ITC experiments performed, the negative binding enthalpy was found to be a negligible quantity. The study highlights one of the first instances of negligible enthalpy change during DNA–ligand interaction. This negligible enthalpy of ctDNA–REV peptide binding could be partially explained by different structural changes that occur in the microenvironment of the complex. Heat capacity changes during REV peptide binding to ctDNA provide information about the thermodynamic mode of binding. Variable temperature ITC experiments were exothermic in nature and slightly affected during temperature increment. The negligible change in heat capacity (ΔC_p = 0.007 kcal mol⁻¹ K⁻¹) is commonly not seen in this type of interaction. Such cases are unusual in calorimetric-based experiments and this finding is the first report for a DNA–peptide interaction. The negligible change in heat capacity is due to electrostatic interaction and DNA condensation in the presence of a cationic peptide. The contribution of different thermodynamic parameters towards series of temperature and correlation of ΔH, ΔS, and ΔG together is indicative of entropy-based binding phenomenon (Figure 7).

The observed positive values of ΔS and negative values of ΔH are consistent with the characteristics of van der Waals, hydrophobic, and electrostatic interactions. The change in entropy during binding of REV peptide and ctDNA is mainly due to change in structure, conformation, orientation, number of water molecules, and ions released. The CD results also show that REV peptide binding accompanies large conformational changes of ctDNA resulting in B → Ψ transition. Unfavorable entropy changes during ctDNA–REV peptide binding were not observed. The small and low enthalpy change during ITC experiment could be due to desolvation and deionization of polar groups, which negatively compensates for the energy released due to hydrogen bond formation. Therefore, REV peptide can be considered as minor-groove binding ligand which gives exothermic binding enthalpies. Because REV is a cationic peptide, it can charge-neutralize and condense ctDNA to form compact toroids or rod type structures.

REV peptide is composed of 11 positively charged Arg residues and 2 negatively charged residues (Asp and Glu). The computed overall hydrophobicity of REV peptide is −2.791 (http://web.expasy.org/protparam/), thus confirming the hydrophilic nature of the peptide. The highly charged amino acids within peptide confer the tendency to get highly solvated in the aqueous solution. The packing of ctDNA–REV peptide is due to remaining interfacial water molecules and stabilizing of the complex is due to presence of hydrogen bond network between polar bases of ctDNA and charged amide groups of REV peptide. The positive and favorable entropic change in ITC during the ctDNA–REV peptide complex formation indicates either the sequestration of water molecules around the ctDNA–REV peptide complex or a more structured complex as compared to native ctDNA. The positive ΔS values could arise due to hydrophobic effects and intermolecular hydrogen bonds network. The negative and negligible change in enthalpy suggests the involvement of ionic, dipole moment, van der Waals interaction, and the presence of hydrogen bonds. It appears that the polar arginine residues (Arg 3, Arg 6, 7, Arg 9–12, Arg 14, Arg 16, Arg 18, and Arg 22) of REV peptide makes hydrogen bonds or electrostatically interact with the polar phosphate backbone of ctDNA resulting in burial of polar groups leading to the reduced solvent accessibility to the complex.

These results clearly indicate that the binding between ctDNA and REV peptide is a specific electrostatic interaction governed by oppositely charged molecules. It is possible that three modes of electrostatic interactions occur during ctDNA–REV peptide binding phenomena. First, the electrostatic interaction with the negative charged nucleic sugar phosphate structure binds with positively charged REV peptide. Second, the binding interaction with two major grooves of the ctDNA double helix and the third mode is the intercalation with stacked base pairs of native ctDNA macromolecule.