Biophysical Characterization and Molecular Docking Studies of

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Article pubs.acs.org/Biomac

Biophysical Characterization and Molecular Docking Studies of Imidazolium Based Polyelectrolytes−DNA Complexes: Role of Hydrophobicity Kasina Manojkumar,† K. T. Prabhu Charan,† Akella Sivaramakrishna,† Prakash C. Jha,‡ Vijay M. Khedkar,§ Ramamoorthy Siva,∥ Gurunathan Jayaraman,⊥ and Kari Vijayakrishna*,† †

Organic Chemistry Division, School of Advanced Sciences, ∥Plant Biotechnology Division, School of Biosciences and Technology, and ⊥Bioinformatics Division, School of Biosciences and Technology, VIT University, Vellore-632014, Tamil Nadu, India ‡ School of Chemical Sciences, Central University of Gujarat, Sector-30, Gandhinagar-38200, Gujarat, India § Combi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune 411008, Maharashtra, India S Supporting Information *

ABSTRACT: Nonviral gene delivery vectors are acquiring greater attention in the field of gene therapy by replacing the biological viral vectors. DNA− cationic polymer complexes are one of the most promising systems to find application in gene therapy. Hence, a complete insight of their biophysical characterization and binding energy profile is important in understanding the mechanism involved in nonviral gene therapy. In this investigation, the interaction between calf thymus DNA (ctDNA) and imidazolium-based poly(ionic liquids) (PILs) also known as polyelectrolytes with three different alkyl side chains (ethyl, butyl, and hexyl) in physiological conditions using various spectroscopic experiments with constant DNA concentration and varying polyelectrolyte concentrations is reported. UV− visible absorption, fluorescence quenching studies, gel electrophoresis, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) have confirmed the binding of polyelectrolytes with DNA. UV−vis absorption measurements and fluorescence quenching revealed that the binding between DNA and the polyelectrolyte is dominated by electrostatic interactions. Additionally, CD and FTIR results indicated that the DNA retained its B-form with minor perturbation in the phosphate backbone without significant change in the conformation of its base pairs. Preference for alkyl side chains (KPIL‑Ethyl Br < KPIL‑Butyl Br < KPIL‑Hexyl Br) toward efficient binding between the polyelectrolyte and DNA was inferred from the binding and quenching constants calculated from the absorption and emission spectra, respectively. Further, in silico molecular docking studies not only validated the observed binding trend but also provided insight into the binding mode of the polyelectrolyte−DNA complex.

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INTRODUCTION Gene therapy, a pivotal method involving the transfer of genetic material into target cells in order to rectify the expression of a particular gene, has attracted significant interest throughout the research community in the past few decades for curing genetic diseases from inherited disorders to cancers.1,2 The development of safe, efficient, and biocompatible gene delivery vectors has become a crucial challenge in the field of gene therapy and clinical applications. Viral vectors are found to be potential gene transfer vehicles but acquired severe drawbacks causing immune reactions, mutations, and cancer in patients.3 These safety concerns have encouraged the researchers toward the development of nonviral vectors possessing minimal mutagenesis.4 These synthetic vectors which mostly include cationic polymers and cationic lipids have gained much popularity over their counterparts and emerged as a recent topic of interest in the field of gene therapy.5−9 The DNA−cationic polymer complexes involving electrostatic interactions between the © 2015 American Chemical Society

cationic polymeric chains and negatively charged phosphate backbone of DNA are being developed as promising nonviral gene delivery systems due to their salient features such as aqueous solubility, charge neutralization, pH sensitivity, and tunable physiochemical properties.10−14 Several cationic polymers such as linear poly(amidoamine)s (PAA),15 poly[2(dimethylamino)ethyl methacrylate],16,17 poly(3-guanidinopropyl methacrylate),18 ester-functionalized linear poly(ethylenimine),19 polyhistidine,20 poly(ethylenimine), and poly(L-lysine)21 and triazole based click polymers22 have been employed so far to study their interaction with DNA. Their transfection efficiency and cell viability were also evaluated and comprehended with their DNA binding characteristics. Received: December 14, 2014 Revised: February 10, 2015 Published: February 11, 2015 894

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recrystallization. All other purchased chemicals were of analytical grade and used as such without further purification. The imidazolium-based ionic liquids (IL-Ethyl Br, IL-Butyl Br, and IL-Hexyl Br) were synthesized according to the literature report.44 10 mM TRIS-HCl buffer was prepared using double distilled deionized water, and the pH was adjusted to 7.4 using 0.1 M NaOH solution. 1% w/w of ctDNA in 10 mM TRIS-HCl buffer (pH 7.4) was prepared with occasional stirring and stored in a refrigerator at 5 °C. The final concentration of ctDNA was determined by UV−visible absorption spectroscopy at 260 nm using known molar extinction coefficient, ε260 = 6600 M−1 cm−1 (expressed as molarity of phosphate groups). The absorbance of a dilute solution of ctDNA in buffer solution at 260 nm was recorded to be 0.42091, the concentration, calculated using ε260, was found to be 63.8 μM, and the final concentration of ctDNA stock solution was estimated to be 12.7 mM. The ctDNA used for all the experiments was sufficiently free of protein with the ratio of UV absorbance of ctDNA in buffer solution at 260 and 280 nm (A260/A280) ≈ 1.8.45 General Procedure for the Preparation of Polyelectrolytes (PIL-Alkyl Br). Imidazolium-based polyelectrolytes of the type poly(N-alkyl-3-vinylimidazolium bromide)s were synthesized according to the literature report.28 N-Vinylimidazole was homopolymerized using free radical polymerization with the degree of polymerization targeted as 50 repeating units. The obtained poly(N-vinylimidazole) was purified by precipitation in cold chloroform and then quaternized with an excess of three different alkyl bromides and used for the DNA binding studies after purification. The quaternization of poly(Nvinylimidazole) with different alkyl halides were followed by 1H NMR, and the relative integration confirm the quantitative conversions as shown in Figure S1 (see the Supporting Information). The hydrophilic polyelectrolytes PIL-Alkyl Br were converted to hydrophobic PILAlkyl NTf2 by simple anionic metathesis using LiNTf2. Molecular weights of these PIL-Alkyl NTf2 polymers were calculated by Waters gel permission chromatography instrument with Styragel columns equipped with an RI detector in THF solvent with a flow rate of 1 mL/min using polystyrene internal standards. The molecular weights, Mn (polydispersity index, PDI), of [PIL-Ethyl NTf2], [PIL-Butyl NTf2], and [PIL-Hexyl NTf2] were found to be 20,800 (1.40), 24,400 (1.56), and 29,950 (1.64), respectively as shown in Figure S2 (see the Supporting Information).

On the other hand, PILs containing a polymerized vinylic backbone and an ionic moiety quarternized at the heteroatom with hydrophobic alkyl groups possess unique properties of ionic liquids (ILs) as well as polymers.23−25 The potential and innovative applications of polyelectrolytes include biosensors, catalytic membranes, absorbing membranes, support for catalysts, polymeric surfactants, electrochemical devices, solid phase microextraction, and phase transfer medium.23,26−28 These polyelectrolytes are advancing as an interesting class of cationic polymers having significant binding affinity to DNA. So far in the literature, the interaction of phosphonium,29 sulfonium,30 ammonium,31,32 and pyridinium33,34 based polyelectrolytes with DNA has been reported, and their binding mode and transfection efficiency has been discussed. To date, very little data are available in literature regarding the interaction between imidazolium-based polyelectrolytes and DNA. Recently, Long et al. reported the DNA binding studies of imidazolium-based copolymer quaternized with hydroxylfunctionalized alkyl chains.35 The authors have discussed the contribution of charge density and hydroxyl concentration on DNA binding, cytotoxicity, and in vitro transfection efficiency. It was found that DNA binding increased with the increase in charge density and hydroxy levels, whereas a systematic change in both parameters had a significant impact on transfection efficiency. However, a detailed biophysical characterization, structure− activity relationship, and role of alkyl side chain’s hydrophobicity on the interactions between imidazolium-based polyelectrolytes and DNA are still unclear. We hypothesize that the tuning of the polyelectrolyte’s hydrophobicity may be of help in studying the role of hydrophobic interaction between the polyelectrolyte’s alkyl side chains and DNA, apart from the electrostatic interaction between cationic polymer moieties and phosphate anions of DNA. Moreover, there are several reports36−39 in the literature which has provided the evidence for the important role of cationic polymer’s hydrophobicity in gene delivery where it was found to enhance the DNA condensation,40,41 promote the polymer−DNA complex interaction with cell membranes,40 and facilitate the release of nucleic acid from the polymeric carriers. 42 Also, the incorporation or increase in hydrophobic alkyl chains in the cationic polymer was found to increase the binding with DNA.43 In this study, imidazolium-based polyelectrolytes with different alkyl side chain lengths (ethyl, butyl, and hexyl) were synthesized, and their electrostatic and hydrophobic interactions with DNA were examined. For the first time to our knowledge, the binding characteristics, mode of interaction, and the effect of imidazolium-based polyelectrolyte alkyl side chains hydrophobicity on the binding affinity to DNA were investigated in detail using different experiments such as UV−visible absorption, fluorescence, CD, agarose gel electrophoresis, and FTIR spectroscopy. In addition, molecular docking studies were carried out to visualize the binding mode and preferential docking position of polyelectrolytes in DNA. Furthermore, the structure−activity relationship was explained by comparing the binding affinity of polyelectrolytes with DNA.

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UV−Visible Absorption. The absorption spectra were recorded on a HITACHI U-2910 UV−visible spectrophotometer with 1 cm quartz cuvette at 298.15 ± 0.15 K. The absorbance was measured at pH 7.4 by keeping the DNA concentration constant (63.5 μM) and varying the polyelectrolyte concentration (0, 0.375, 0.75, 1.25, and 1.6 μM). The solutions were incubated for 5 min and then scanned at a wavelength between 200 and 400 nm. The interference due to absorbance of the polyelectrolyte was subtracted during the calculation of absorbance of the DNA−polyelectrolyte complex. Fluorescence Quenching Studies. The fluorescence quenching studies of ethidium bromide (EtBr)-bound ctDNA were carried out using the HITACHI F-700 fluorescence instrument. The ctDNA at 254 μM concentration and the EtBr at 25.3 μM concentration were prepared in 10 mM TRIS-HCl buffer (pH 7.4) and used in the experiment. The EtBr−DNA complex was titrated using different concentrations (0, 0.25, 0.75, 1.5, 2.5, 4, and 6 μM) of the polyelectrolyte. The solutions were gently mixed and incubated for 5 min, and then the readings recorded at 298.15 ± 0.15 K. Salt Effect. The effect of ionic strength on the fluorescence quenching of the EtBr−DNA complex by the polyelectrolyte was studied using various concentrations of NaCl (0.2 M, 0.6 M, and 0.8 M). The experiment was performed using 254 μM concentration of ctDNA, 25.3 μM concentration of EtBr, and different concentrations (0.25, 0.45, 0.75, 1, 1.25, 1.45, 1.75, 2, 2.25, and 2.45 μM) of PIL-Ethyl Br in the presence of NaCl.

EXPERIMENTAL SECTION

Materials. Alkyl halides, such as, n-bromoethane, n-bromobutane, and n-bromohexane; organic solvents; ethidium bromide; TRIS-HCl; NaOH; N-vinylimidazole; AIBN; and sodium salt of calf thymus DNA (ctDNA) were purchased from Sigma-Aldrich. AIBN was used after 895

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Biomacromolecules Circular Dichroism. Circular dichroism experiments were performed on a JASCO J-715 spectropolarimeter, and the spectra were recorded using a 1 cm path length rectangular quartz cuvette at 298.15 ± 0.15 K. The circular dichroism titrations were performed using a fixed concentration of ctDNA (635 μM) and various concentrations (0, 0.16, 0.33, 0.5, 0.66, 1.0, and 1.3 μM) of the polyelectrolyte prepared in 10 mM Tris-HCl buffer and scanned at a wavelength between 220 and 320 nm, bandwidth of 1 nm, response time of 1 s, and an average of three scans to reduce the signal-to-noise ratio. The baseline of the ctDNA−polyelectrolyte complexes CD spectra was corrected by subtracting with that of 10 mM TRIS-HCl buffer, and the experiment was carried out in an atmosphere of nitrogen. Agarose Gel Electrophoresis. The electrophoretic mobility of the ctDNA−polyelectrolyte complexes at varying concentrations of polyelectrolytes was determined by gel electrophoresis using 1% agarose gel in a buffer containing 45 mM TRIS-Borate buffer and 1 mM EDTA buffer (TBE). The ctDNA−polyelectrolyte complexes were prepared by mixing an appropriate amount of 12.7 mM DNA and various concentrations of polyelectrolytes (0, 0.1, 0.3, 0.5, 0.9, and 1.1 mM) and allowed to incubate for 30 min at 25 °C. These solutions were placed carefully inside the wells of 1% agarose gel along with the gel loading dye to visualize the gel run. The gel was run at 100 V for 45 min, and then the DNA was visualized under the UV transilluminator after staining the gels with ethidium bromide overnight at 25 °C. The images of DNA were obtained using gel documentation instrument. FTIR Spectroscopy. FTIR spectra of ctDNA and its complexes with the polyelectrolyte were recorded with a SHIMADZU-IR AFFINITY-1 spectrophotometer equipped with a DLATGS (deuterated L-alanine triglycene sulfate) detector and a KBr beam splitter. The ctDNA−polyelectrolyte complexes were made using 10 mM TRIS-HCl buffer solution to give the desired polyelectrolyte/ctDNA ratios of 1/25, 1/10, and 1/5 at the final ctDNA concentration of 12.7 mM. The prepared solutions were incubated for 30 min at 25 °C and then recorded using KBr pellets. Molecular Docking. Molecular docking studies were carried out using the Glide (Grid-Based Ligand Docking With Energetics)46,47 program incorporated in the Schrodinger molecular modeling package (Schrodinger, Inc., USA) which is an interactive molecular graphics program for the interaction, docking calculations, and identification of the possible binding site of the biomolecules.48,49 The receptor, DNA duplex (PDB ID 425D) retrieved from Protein Data Bank (www.rcsb.org) contained 12 base pairs.50 The B-DNA structure was optimized using the Protein Preparation Wizard. The B-DNA crystal structure was preprocessed by deleting the crystallographically observed water molecules (water without H bonds) as no water molecule was found to be conserved and optimizing the hydrogen bonds. The hydrogen atoms were added to the DNA structure corresponding to pH 7.0. After assigning the charge and protonation state, energy minimization with root-meansquare deviation (RMSD) value of 0.30 Å was carried out using Optimized Potentials for Liquid Simulations (OPLS-2005) force field. On the other hand, the 3D geometries of polyelectrolytes (pentamer) were optimized using the LigPrep module, and the partial charges were ascribed using the OPLS2005 force-field as performed in Glide. The binding pocket of B-DNA is defined by a 14 × 14 × 14 Å box that is centered on the geometric centroid of the B-DNA structure. Sufficiently

large grids were chosen to include a significant part of B-DNA. The extra-precision scoring function in Glide was used to rank the docking poses and measure the binding affinity of polyelectrolytes (pentamer) to B-DNA.46,47

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RESULTS AND DISCUSSION Poly(N-alkyl-3-vinylimidazolium bromide)s with three different alkyl side chains (ethyl, butyl, and hexyl) were synthesized by quaternizing the radically synthesized poly(N-vinylimidazole) according to the literature report (Scheme 1).28 Since, the Scheme 1. Imidazolium-Based Poly(ionic liquids) Used in This Study

backbone of all the employed polymers (PIL-Ethyl Br, PILButyl Br, and PIL-Hexyl Br) of this study are from the same unit, i.e. poly(N-vinylimidazole), it facilitates a comparative study with the respective alkyl side chain length.

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DNA BINDING STUDIES The thermodynamic parameters and electrostatic interactions between ionic liquids of the type N-alkyl-3-methylimidazoliums and DNA were already reported in the literature.51−54 As a preliminary investigation, we have studied the fluorescence quenching of the EtBr−DNA complex using N-hexyl-3methylimidazolium bromide (IL-Hexyl Br) (Figure S3). Surprisingly, IL-Hexyl Br was found to quench the fluorescence only at high concentrations (0.1 to 1.7 mM), whereas PILHexyl Br showed significant fluorescence quenching at minute concentrations (0.25 to 6 μM), and the fluorescence quenching constants are as follows: KIL‑Hexyl Br = 0.0742 × 103 M−1 and KPIL‑Hexyl Br = 0.5926 × 106 M−1 (Figure S4). Moreover, the relative binding energy from the molecular docking studies between ILs and DNA was found to be less than that of polyelectrolytes. (The detailed docking modes (Figure S5) and docking studies for ILs (IL-Ethyl Br, IL-Butyl Br, and ILHexyl Br) and DNA are given in the Supporting Information.) Hence, these findings have encouraged us to continue the DNA binding studies with imidazolium-based polyelectrolytes. UV−Visible Absorption. The interaction between polyelectrolytes and ctDNA was examined by UV−vis absorption spectra of ctDNA with varying concentrations of polyelectrolytes (0.375 to 1.6 μM), which has been illustrated in Figure 1. The absorption spectra of ctDNA showed an increase in absorbance at 260 nm with the gradual addition of polyelectrolytes. This hyperchromic shift is ascribed to the electrostatic mode of interaction between the cationic imidazolium groups of polyelectrolytes and phosphate anions of ctDNA.55 The absorbance of ctDNA increased dramatically with 0.375 and 0.75 μM of the polyelectrolyte, and a negligible or minimal increase was noted with 1.25 and 1.6 μM of the polyelectrolyte. These observations concurred well with the DNA binding studies of cationic polymers available in the literature.56,57 It is assumed that the interaction between DNA and a ligand (polyelectrolyte) is 1:1, and the equilibrium 896

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Figure 1. UV−vis absorption spectra of free ctDNA (63.5 μM) and ctDNA−polyelectrolyte complexes with different concentrations (0, 0.375, 0.75, 1.25, and 1.6 μM) of the polyelectrolyte (i) PIL-Ethyl Br, (ii) PIL-Butyl Br, and (iii) PIL-Hexyl Br.

Figure 2. Fluorescence emission spectra of the EtBr−ctDNA complex with the addition of different concentrations (0, 0.25, 0.75, 1.5, 2.5, 4, and 6 μM) of (i) PIL-Ethyl Br, (ii) PIL-Butyl Br, and (iii) PIL-Hexyl Br. The comparative graph (iv) represents the decrease in relative fluorescence intensity of the EtBr−ctDNA complex with an increase in the alkyl side chain length of polyelectrolytes.

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KPIL‑Butyl Br = 0.3167 × 106 M−1, and KPIL‑Hexyl Br = 0.5926 × 106 M−1 are the calculated values of polyelectrolyte quenching constants. This effect of alkyl side chain length on the quenching of fluorescence intensity may be attributed to the increase in hydrophobic interactions between polyelectrolyte alkyl side chains and ctDNA along with the electrostatic interactions.53 This trend is in agreement with the reported literature regarding binding studies between ctDNA and ILs with different alkyl side chains.54 Salt Effect. Monitoring the spectral changes with varying ionic strength is an efficient method to identify the binding mode between molecules and DNA.63 The addition of NaCl would weaken the electrostatic interactions between phosphate backbone of DNA and cationic molecules due to their competition with sodium ions. The emission spectra of the EtBr−DNA complex with the addition of the polyelectrolyte in the presence of varying concentrations of NaCl (0.2, 0.6, and 0.8 M) were monitored in order to evaluate the contribution of electrostatic interaction in the binding between ctDNA and polyelectrolyte. PIL-Ethyl Br was used as an example, and the emission spectra were represented in Figure S6 (see the Supporting Information). As shown in Figure 3, the

between them could be established according to the following relationship:58,59 DNA + Ligand ↔ DNA: Ligand

K=

[DNA: Ligand] [DNA][Ligand]

The binding constants of polyelectrolytes were calculated according to the literature reports.58−61 A double reciprocal plot of 1/A−A0 and 1/CPolyelectrolyte is linear, and the binding constant, K, was calculated from the ratio of the intercept to the slope. A0 and A correspond to the absorbance of free ctDNA at 260 nm in the absence of the polyelectrolyte and absorbance recorded with the addition of different concentrations of the polyelectrolyte, respectively. The graphs (Figure 1) followed linear regression, and the binding constants increased with the increase in the alkyl chain length of the polyelectrolytes thereby suggesting the contribution of alkyl chain’s hydrophobicity in the interaction between the polyelectrolyte and DNA. The estimated binding constants of polyelectrolytes are KPIL‑Ethyl Br = 1.6008 × 106 M−1, KPIL‑Butyl Br = 1.6493 × 106 M−1, and KPIL‑Hexyl Br = 3.2087 × 106 M−1. Fluorescence Quenching Studies. EtBr is a dye that binds to ctDNA by intercalation with DNA bases and is generally used as a fluorescent probe in DNA binding studies. In this experiment, EtBr was used to study the interactions between ctDNA and the polyelectrolyte through fluorescent quenching studies. The fluorescence intensity of the EtBr− ctDNA complex decreases when ctDNA gets condensed or compacted by the quencher (competing molecule) thereby restricting the intercalation with EtBr.21 In this process, EtBr is leached out freely into the bulk solvent and, therefore, decreases the fluorescence intensity of the complex. It is expected that the polyelectrolyte would condense the ctDNA and form compact macromolecular structures through electrostatic interaction between polymeric imidazolium cations and phosphate anions of ctDNA, resulting in insufficient space for intercalation with EtBr. The emission spectra of the EtBr− ctDNA complex with varying concentrations of polyelectrolytes are represented in Figure 2. The fluorescence intensity of the EtBr−ctDNA complex decreased concomitantly with the addition of increasing concentrations (0.25 to 6 μM) of polyelectrolytes indicative of fluorescence quenching by the polyelectrolytes employed. The fluorescence intensity of the EtBr−DNA complex decreased steadily from 0.25 to 2.5 μM of the polyelectrolyte and then decreased abruptly from 4 to 6 μM of the polyelectrolyte. The fluorescence quenching constant, KSV, was calculated using the following Stern−Volmer equation:62

Figure 3. Graph representing the decrease in relative fluorescence intensity of EtBr bound ctDNA by polyelectrolyte with an increase in the ionic strength.

fluorescence quenching of the EtBr−DNA complex by the polyelectrolyte decreased drastically with the increase in NaCl concentration, which is ascribed to the competition between cationic imidazolium moiety of the polyelectrolyte and sodium ions. The fluorescence quenching constants of the polyelectrolyte also decreased with the increase in the ionic strength suggesting the strong dependence of fluorescence intensity on ionic strength and the sequence is as follows: K0 M = 0.1455 × 106 M−1 > K0.2 M NaCl = 0.1010 × 106 M−1 > K0.6 M NaCl = 0.04828 × 106 M−1 > K0.8 M NaCl = 0.03510 × 106 M−1. The results clearly confirm the mode of interaction between DNA and polyelectrolytes as mainly electrostatic.56 Circular Dichroism. The conformational changes in the secondary structure of DNA before and after the addition of polyelectrolytes were studied using a CD spectropolarimeter. The CD spectra of ctDNA after the addition of varying concentrations (0.16 to 1.3 μM) of polyelectrolytes are shown in Figure 4. The characteristic elliptic peaks at 247 nm (negative) and 275 nm (positive) are caused due to π−π base stacking and helicity of ctDNA, respectively. This further suggests that the ctDNA retained the B-form after binding with polyelectrolytes.64 On the other hand, the employed polyelectrolytes do not have any optical activity in the present investigation. The molar ellipticity progressively decreased at

F0 = 1 + KSV(8) F

Here, F0 is the steady-state fluorescence intensity of the EtBr−DNA complex in the absence of the polyelectrolyte, F is the intensity of the EtBr−DNA complex in the presence of the polyelectrolyte (quencher), [8 ] is the concentration of quencher, and KSV was obtained from the slope of [8 ] vs F0/F plot. The graphs (Figure 2d) follow an almost linear regression, and the quenching constant increases with the increase in the polyelectrolyte’s alkyl side chain length, which is inconsistent with the binding trend observed in UV−vis absorption spectral results. KPIL‑Ethyl Br = 0.2676 × 106 M−1, 898

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Figure 4. CD spectra of DNA (635 μM) with different concentrations (0, 0.16, 0.33, 0.5, 0.66, 1.0, and 1.3 μM) of polyelectrolytes (a) PIL-Ethyl Br, (b) PIL-Butyl Br, and (c) PIL-Hexyl Br.

Figure 5. Electrophoretic mobility of the ctDNA−polyelectrolyte complexes [(a) ctDNA - PIL-Ethyl Br (b) ctDNA - PIL-Butyl Br (c) ctDNA - PILHexyl Br] on 1% agarose gel. Lanes 0, 1, 2, 3, 4, 5, and 6 correspond to the ctDNA−polyelectrolyte complexes with 12.7 mM DNA and 0, 0.1, 0.3, 0.5, 0.9, and 1.1 mM concentrations of the polyelectrolyte, respectively.

further supported the results discussed previously in the UV− vis absorption and fluorescence studies. Agarose Gel Electrophoresis. Gel electrophoresis allows the visualization of the interaction between ctDNA and the polyelectrolyte. The representative gel images of the ctDNA− polyelectrolyte complexes with different concentrations and alkyl side chains of polyelectrolytes are depicted in Figure 5. Lane 0 represents the illumination of free ctDNA, and lanes 1− 6 in Figure 5a, b and lanes 1−4 in Figure 5c represent the illumination of incompletely neutralized ctDNA migrating in the electric field toward the anode. The illumination decreased with the increase in the concentration and alkyl side chain length of the polyelectrolyte, thus indicating the formation of the ctDNA−polyelectrolyte complexes. No illumination was observed in lanes 5 and 6 in Figure 5c showing complete neutralization of anionic phosphate groups of ctDNA with cationic imidazolium groups of the polyelectrolyte. From the gel electrophoresis, it could be summarized that the ctDNA− PIL-Hexyl Br complex showed complete neutralization with no

both positive (247 nm) and negative (275 nm) bands with an increase in the concentration of polyelectrolytes. This shift at both the positive and negative bands indicated an electrostatic mode of interaction between the phosphate backbone of DNA and cationic imidazolium groups of polyelectrolytes that is in accordance with UV−vis absorption and salt effect results. Moreover, a slight red shift was observed at the crossover region in all the CD spectra of ctDNA bound to polyelectrolytes. Also, the decrease in the intensity of CD signals with an increase in the concentration of the polyelectrolyte may be due to the condensation of ctDNA by the polyelectrolyte.65 From the CD results, it is evident that there are no significant alterations in the secondary structure of DNA after binding to polyelectrolytes, and the DNA’s helicity was well retained. Though the change in molar ellipticity at 247 nm did not follow a regular trend with respect to alkyl chains, the ellipticity at 275 nm decreased appreciably with the increase in the polyelectrolyte’s alkyl side chain length as shown in Figure S7a and b (see the Supporting Information). This trend 899

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Figure 6. Stacked view of FTIR spectra of the polyelectrolyte/ctDNA complexes in the molar ratios of 1/25, 1/10, and 1/5 in the region of 1800 to 800 cm−1.

Figure 7. Binding mode of pentamer of polyelectrolytes (a) (PIL-Ethyl Br)5, (b) (PIL-Butyl Br)5, and (c) (PIL-Hexyl Br)5 with B-DNA.

A prominent shift in phosphate asymmetric stretching from 1083 to 1087 cm−1 was seen at 1/10 and 1/5 molar ratios of the polyelectrolyte (PIL-Ethyl Br, PIL-Butyl Br, and PILHexyl Br); whereas, the shift from 1083 to 1085 cm−1 was noted at 1/25 molar ratio of PIL-Butyl Br and PIL-Hexyl Br. Also, phosphate bending at 990 cm−1 shifted slightly to 989 cm−1 at all the molar ratios of all three polyelectrolytes employed, and the shifts of bands are close to the experimental error of the equipment. It may be concluded that there was no considerable change in the bands of DNA base pairs, whereas the contrary was observed in the phosphate asymmetric bands implying an electrostatic interaction between the oppositely charged groups of polyelectrolytes and DNA. Molecular Docking. In an effort to understand and interpret the molecular mechanism of the interaction between polyelectrolytes with DNA, molecular docking was performed to simulate the modes of interactions between the polyelec-

illumination in 5 and 6 lanes as seen in Figure 5c; whereas ctDNA−PIL-Ethyl Br and ctDNA−PIL-Butyl Br complexes showed partial neutralization. FTIR Spectroscopy. FTIR spectra of free ctDNA and ctDNA−polyelectrolyte complexes with different molar ratios of the polyelectrolyte are represented in Figure 6. The vibrational bands of free ctDNA at 1261, 1402, and 1639 cm−1 are assigned to thymidine, cytosine, and adenine bases, respectively, according to the literature reports.66−69 Bands at 1087 and 991 cm−1 denote symmetric stretching and bending frequencies of phosphate groups, respectively.69 After the addition of the polyelectrolyte to ctDNA, no characteristic change was observed in DNA base-pair bands (thymidine, 1261 cm−1 and adenine, 1639 cm−1), except for a slight shift in the cytosine band from 1402 to 1400 cm−1 at 1/10 molar ratio of polyelectrolyte/ctDNA implying no considerable intercalative mode of interaction between polyelectrolyte and ctDNA bases. 900

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Biomacromolecules trolytes with five repeating monomeric units and B-DNA. Structures of the polyelectrolyte and DNA were kept flexible to attain different conformations in order to predict the best docked complex. The study revealed that groove binding makes intimate contacts with the walls of the groove, and as a result of this interaction, numerous electrostatic and van der Waals interactions occur between the units of the polyelectrolyte and DNA bases and its phosphate backbone. From the ensuing docked structures, it is clear that all the polyelectrolytes fit snugly into the curved contour of the targeted DNA in the minor groove, with the walls of the groove in close contact with imidazolium scaffold and associated alkyl side chains stabilized by strong electrostatic interaction (Figure 7). Relative binding energy docking scores of the docked polymeric structures was found to be −74.66 kcal/mol (−10.56), −83.08 kcal/mol (−11.67), and −90.91 kcal/mol (−13.32) for pentamers (PILEthyl Br)5, (PIL-Butyl Br)5, and (PIL-Hexyl Br)5, respectively. The binding energy of PIL-Ethyl Br and PIL-Butyl Br was found to be lower than that of PIL-Hexyl Br. Higher negative binding energy indicates a more stable combination with DNA. While a consistent interaction was observed between the imidazolium nucleus and the DNA base pairs across the three polyelectrolytes, it was the varying alkyl chain length that contributed to the variation in the binding strengths of these polyelectrolytes. Thus, it is concluded that the binding ability of PIL-Hexyl Br with DNA is stronger than that of PIL-Ethyl Br and PIL-Butyl Br. The docking scores and the corresponding binding energy values revealed an increase in polyelectrolytes binding affinity with an increase in their alkyl side chain length (KPIL‑Ethyl Br > KPIL‑Butyl Br > KPIL‑Hexyl Br). Furthermore, analysis of the binding modes of these polyelectrolytes revealed that the B-form was well retained by the DNA with minor perturbation in its phosphate backbone and no significant change in the base pairs after complexation with polyelectrolytes, which is in good agreement with the experimental observations.

groove binding with DNA along with predominant electrostatic interaction between the oppositely charged groups.

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ASSOCIATED CONTENT

* Supporting Information S

Results of fluorescence quenching of the EtBr−DNA complex using IL-Hexyl Br and the detailed docking studies for ILs (ILEthyl Br, IL-Butyl Br, and IL-Hexyl Br) and DNA. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 91 416 224 2334. Fax: 91 416224 3092. E-mail: kari@ vit.ac.in. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS V. Kari thanks DST-SERB, India (Project NO: SR/S1/OC-22/ 2012) for the financial support. The authors also thank DSTVIT-FIST for NMR and VIT-SIF for other instrumentation facilities.

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REFERENCES

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CONCLUSION Imidazolium-based polyelectrolytes with three different alkyl side chains (ethyl, butyl, and hexyl) derived radically were successfully employed to study their interaction with ctDNA. The absorption spectra of ctDNA showed a hyperchromic shift with the increase in polyelectrolyte concentration that may be ascribed to the electrostatic interaction between the cationic imidazolium groups of the polyelectrolyte and phosphate anions of ctDNA. This electrostatic mode of interaction was further confirmed by the effect of ionic strength on the binding of the polyelectrolyte with ctDNA. The infrared and CD spectroscopic results showed evidence that the DNA remained in the B-form without much perturbation to the secondary structure after binding to polyelectrolytes. The binding constants of polyelectrolytes obtained from the absorption spectra followed the same trend of KPIL‑Ethyl Br < KPIL‑Butyl Br < KPIL‑Hexyl Br as that of the quenching constants calculated from the emission spectra. This binding trend demonstrates an increase in binding between polyelectrolytes and DNA with an increase in the polyelectrolyte’s alkyl side chain length suggesting the contribution of hydrophobic interaction between the polyelectrolyte’s alkyl side chains and DNA apart from the electrostatic interaction. The above binding trend was further confirmed by molecular docking studies with respect to the binding energy of polyelectrolytes toward DNA. Furthermore, it was visualized that polyelectrolytes (pentamer) showed 901

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