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Spectroscopic and Molecular Docking Study of the Interaction of DNA with a Morpholinium Ionic Liquid Ashok Pabbathi and Anunay Samanta* School of Chemistry, University of Hyderabad, Hyderabad 500046, India S Supporting Information *

ABSTRACT: The structural integrity of a nucleic acid under various conditions determines its utility in biocatalysis and biotechnology. Exploration of the ionic liquids (ILs) for extraction of DNA and other nucleic acid based applications requires an understanding of the nature of interaction between the IL and DNA. Considering these aspects, we have studied the interaction between calf-thymus DNA and a less toxic morpholinium IL, [Mor1,2][Br], employing fluorescence correlation spectroscopy (FCS), conventional steady state and time-resolved fluorescence, circular dichroism (CD) and molecular docking techniques. While the CD spectra indicate the stability of DNA and retention of its B-form in the presence of the morpholinium IL, the docking study reveals that [Mor1,2]+ binds to the minor groove of DNA with a binding energy of −4.57 kcal mol−1. The groove binding of the cationic component of the IL is corroborated by the steady state fluorescence data, which indicated displacement of a known minor groove binder, DAPI, from its DNA-bound state on addition of [Mor1,2][Br]. The FCS measurements show that the hydrodynamic radius of DNA remains more or less constant in the presence of [Mor1,2][Br], thus suggesting that the structure of DNA is retained in the presence of the IL. DNA melting experiments show that the thermal stability of DNA is enhanced in the presence of morpholinium IL.

1.0. INTRODUCTION

Chart 1. Structure of Typical Cations and Anions of Ionic Liquids

DNA, the most important biomolecule, which encodes genetic information, finds several applications in material science as nano machines, biosensors, and bone guiding scaffolds.1−3 Recently, DNA-based hybrid catalysts have been found to be quite useful in asymmetric synthesis.4 The long-term storage of DNA, however, is a major challenge, as hydrolytic reactions cause its denaturation when stored in an aqueous environment for a long period.5−7 The stability of DNA also depends on factors such as pH, temperature, and solvent properties.6 To ensure long-term stability of DNA, studies are being conducted in various nonaqueous and mixed solvents and these studies have shown that DNA loses its structure in DMSO and DMF but retains its structure in ethylene glycol.7−9 Most of the reactions involving DNA as a hybrid catalyst are usually carried out in aqueous solution. However, as a large number of organic substrates do not dissolve in aqueous medium, it is often necessary to use nonaqueous or mixed solvent or a novel medium to perform the organic reactions. In recent years, it is shown that the ILs, which possess attractive solvent properties such as high thermal stability, high conductivity, and low vapor pressure and whose physicochemical properties can be tuned simply by changing the ionic constituents (Chart 1), can be used in biocatalyis,10 protein purification and crystallization,11 designing ion conductive DNA films,12,13 and in the separation13 and extraction of DNA.14 Exceptional stability of DNA in the presence of choline © XXXX American Chemical Society

based ILs is attributed to the slow rate of hydrolytic reactions in the presence of ILs.7 Ding et al. studied the interaction between [Bmim][Cl] and DNA using several analytical techniques and concluded that both electrostatic and hydrophobic interactions play important roles in the binding of IL to DNA.15 Pang and co-workers estimated the binding parameters of [Bmim][BF4] with DNA using an electrochemical micromethod.16 Wang and coworkers, who estimated the binding energies of [Cnmim][Br] Special Issue: Biman Bagchi Festschrift Received: March 27, 2015 Revised: May 19, 2015

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DOI: 10.1021/acs.jpcb.5b02939 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B (n = 4, 6, 8, 10, 12) with DNA by monitoring the fluorescence of pyrene, concluded that the electrostatic interaction between DNA and imidazolium ion of the ILs is the dominant factor in the binding.17 Recently, Senapati and co-workers have studied the interaction of [Bmim]+[X]− {X = Cl, NO3, and lactate} and [choline]+[X]− {X = NO3 and lactate} ILs with DNA with the help of molecular dynamics simulation and shown that the cationic component of the ILs binds to DNA grooves and contributes to the stability of DNA in addition to the electrostatic interaction.6 They found that [Bmim]+ binds more effectively to DNA compared to [choline]+ because of its planar geometry and charge delocalization.6 In an earlier study, Fang and co-workers 18 suggested intercalation of the imidazolium cation inside DNA, which is in contrast with the results of Senapati and co-workers. Very recently, molecular dynamics simulation study by Jumbri et al. has shown that imidazolium cations of [Cnmim][Br] (n = 2, 4, 6) ILs bind to the grooves of DNA.19 Considering the fact that research activities involving DNA in ILs have just commenced and there exists significant ambiguities concerning the mode of interaction between the two, it is essential to study this aspect employing varieties of ILs. It is for this reason that we have studied the interaction between calf thymus DNA and [Mor1,2][Br],20 an IL comprising morpholinium cation. Our choice of [Mor1,2][Br] is guided by the fact that it is less toxic (and, hence, more biocompatible) compared to the imidizaloium ILs.21 Specifically, it is reported that [Mor1,2][Br] is nontoxic to B. magna and P. subcapitata.21 We have performed molecular docking studies, steady state and time-resolved fluorescence measurements, fluorescence correlation spectroscopy (FCS) studies, and circular dichroism and DNA melting experiments to obtain a comprehensive picture on the stability of DNA in the presence of this IL and the mode of interaction between the two.

lamp profile was recorded by placing a scatterer (dilute solution of Ludox in water) in place of the sample. The DNA (PDB: 1BNA) crystal structure was obtained from the Protein Data Bank and used for the docking study. The docking study was performed using AutoDock 4.2 software. The ligand structure was optimized using Gaussian 03 (DFT, B3LYP, and 6-31G basis set), and the required file was created through AutoDock software. DNA and ligands were prepared using AutoDock Tools. The grid size was set to 58, 74, and 108 in x-, y-, and z-direction, respectively with a grid spacing of 1.0 Å. All other parameters were default values. The lowest energy docked conformation was selected as the binding mode in each case. A time-resolved confocal fluorescence microscope (MicroTime 200, PicoQuant) was used for the FCS measurements. The excitation source (λexc = 405 nm) was a pulsed diode laser, and it was focused onto the sample using a water immersion objective (60×/1.2 NA). Fluorescence from the sample was collected by the same objective and passed through a dichroic mirror and 430 nm long-pass filter. A pinhole of 50 μm diameter was used for the spatial filtration of signal and passed through a 50/50 beam splitter prior to entering the two singlephoton avalanche diodes (SPADs). The correlation curves were obtained by cross-correlating the signal from two detectors. All FCS measurements were performed with a laser power of 6 μW in aqueous solution (pH 7.4) containing 150 nM DAPI and 50 μM DNA. FCS curves were analyzed using SymphoTime software provided by PicoQuant. The correlation function of the fluorescence intensity is given by23 G (τ ) =

⟨δF(t )δF(t + τ )⟩ ⟨F(t )⟩2

(1)

where ⟨F(t)⟩ is the average fluorescence intensity and δF(t) and δF(t + τ) are the deviations from the mean value at time t and (t + τ) and are given by

2.0. EXPERIMENTAL SECTION 2.1. Materials. DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride), ct-DNA, tris(hydroxymethyl)aminomethane (Tris), and N-methylmorpholine were obtained from SigmaAldrich. Acetone, acetonitrile, and ethyl bromide were procured from Merck. The compound N-ethyl-N-methyl-morpholinium bromide, [Mor1,2][Br], was synthesized according to a previous report,22 and purity was checked by 1H NMR. 1H NMR (400 MHz, D2O, δ/ppm): 4.05−4.00 (s, 4H), 3.60−3.50 (m, 2H), 3.45−3.40 (t, 4H), 3.1 (s, 3H), 1.38−1.30 (t, 3H). 2.2. Instrumentation and Methods. Steady state absorption and fluorescence spectra were recorded on a UV− visible spectrophotometer (PerkinElmer Lambda 35) and a spectrofluorimeter (Fluorolog-3, JobinYvon), respectively. The CD spectra were recorded using a Jasco J-810 spectropolarimeter averaging three scans. The CD, steady state absorption and emission, and time-resolved emission measurements were carried out in aqueous buffered solution (10 mM Tris−HCl, pH 7.4) using a 10 mm path length quartz cuvette. All measurements have been carried out using sample solutions incubated for 24 h. Time-resolved fluorescence measurements were carried out using a time-correlated single-photon counting (TCSPC) spectrometer (Horiba JobinYvon IBH). A PicoBrite diode laser source (λexc = 375 nm) was used as the excitation source and an MCP photomultiplier (Hamamatsu R3809U-50) as the detector. The instrument response function, which was limited by the fwhm of the excitation pulse, was 70 ps. The

δF(t ) = F(t ) − ⟨F(t )⟩, δF(t + τ ) = F(t + τ ) − ⟨F(t )⟩

(2)

The observation volume (0.4 fL) was calibrated using rhodamine 6G (diffusion coefficient 426 μm2 s−1 in water) as described previously.22

3.0. RESULTS AND DISCUSSION 3.1. CD Spectra. The circular dichroism spectra of DNA at various concentrations of [Mor1,2][Br] are shown in Figure 1. The positive band at 275 nm and negative band at 245 nm are the characteristic features of the B-form of DNA due to base stacking and helicity, respectively.24 As can be seen from the figure, upon addition of IL, there are no significant changes in both the positive and negative bands. Small changes of the CD signal amplitude and wavelength at a high concentration of IL, as observed in our case, cannot be attributed to DNA conformational transition, but these must be a reflection of minor changes in base stacking and helicity of DNA.24−26 We have also carried out measurements with samples incubated for different time intervals, and the results do not suggest any time dependent conformational changes of DNA on addition of IL (Figure S1, Supporting Information). It is thus evident that DNA retains its conformation in the presence of [Mor1,2][Br]. The absence of any induced signal in the CD spectra clearly B


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The Journal of Physical Chemistry B

Table 1. Different Interaction Energies (in kcal mol−1) Obtained from Docking Study binding energy

van der Waals + hydrogen bonding + desolvation energy

electrostatic energy

−4.57

−3.68

−1.19

oxygen atoms of thymines, T7 and T19, with bond lengths of 2.69 and 2.79 Å. As stated earlier, the molecular dynamics simulation study by Senapati and co-workers had shown that both imidazolium and choline cations bind to the minor grooves of DNA contributing to the long-term stability of DNA in hydrated ionic liquids.6 A stronger binding of imidazolium cations (ΔGb = −8.53 kcal mol−1 for [Bmim][NO3]) compared to the choline cations (ΔGb = −7.83 kcal mol−1 for [choline][NO3]) was attributed to the planar geometry and charge delocalization in the former that allowed more contact points with DNA bases in the minor groove. The bulky nature of the choline cation was also considered to be partially responsible for its weaker binding. Wang et al. observed that ΔGb of imidazolium ILs (for [Bmim][Br], [Hmim][Br], and [Omim][Br]) with DNA varies between −6.6 and −6.9 kcal mol−1.17 The observation of a somewhat lower ΔGb value of −4.57 kcal mol−1 in our study indicates a weaker binding of morpholinium cation to the DNA grooves compared to imidazolium cations. This result is consistent with the bulky and nonplanar structure of the morpholinium cation compared to the imidazolium cation. As a matter of fact, somewhat weaker binding of the ILs should not be considered as a negative feature, as it can be quite useful in back extraction of DNA into the aqueous phase for its further utilization. It is already shown that back extraction of DNA into the aqueous phase is not a favorable process for imidazolium IL due to its stronger interaction with DNA.14 The stability of DNA depends on the number of water molecules stripped from the DNA due to disruption of the hydration layer by the cations of the ILs. However, the extent of disruption of the hydration shell not only depends on the strength of interactions but also on the penetration power of ions into the shell.6 Clearly, further

Figure 1. CD spectra of ct-DNA (100 μM) in Tris−HCl buffer (10 mM, pH 7.4) with increasing concentrations of [Mor1,2][Br].

suggests that the morpholinium cations do not intercalate into DNA.27 3.2. Molecular Docking. Molecular docking study suggests preferential binding of [Mor1,2]+ to the minor grooves of DNA (Figure 2). This result is consistent with the CD data, which has indicated a negligible change in the conformation of DNA. The binding energy (ΔGb) of this interaction between [Mor1,2]+ and DNA is estimated to be −4.57 kcal mol−1 (Table 1), which corresponds to a binding constant of 2.23 × 103 L mol−1. It can be seen from the table that the contribution of electrostatic interaction to the binding is less compared to van der Waals and hydrogen bonding interactions. Similar observations were made earlier in the case of minor groove binders.28 This study also shows that the cation is located in the AT-rich regions of the minor grooves. Hydrogen bonding interaction between [Mor1,2]+ and the thymine nucleotide of DNA is also evident from the bond lengths (2.10 Å between the C3−H atom of the morpholinium ring and the oxygen atom of the thymine, T8). A three-centered hydrogen bond is also observed involving the C6−H of the morpholinium ring and the

Figure 2. (a) [Mor1,2]+ in the minor groove of DNA and (b) a close-up view of the binding, highlighting the intermolecular H-bonds. C


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The Journal of Physical Chemistry B studies are required to evaluate the effect of morpholinium ILs on the DNA hydration shell. 3.3. Steady State and Time-Resolved Fluorescence. DAPI (Scheme 1) is a frequently used dye molecule, which Scheme 1. Molecular Structure of DAPI

Figure 4. Fluorescence decay profiles (λexc = 375 nm, λem = 510 nm) of DAPI−DNA in buffer (a) and in the presence of 0.5 M (b) and 1.0 M [Mor1,2][Br] (c).

exhibits significant fluorescence enhancement upon binding to the double stranded DNA.29 It is known that DAPI binds to the minor grooves of ct-DNA.29 We have monitored the fluorescence of DNA-bound DAPI (indicated as DAPI− DNA) as a function of the concentration of [Mor1,2][Br] using a solution of 3 μM DAPI and 60 μM DNA. On the basis of earlier reports, we ensure complete complexation of DAPI with DNA under this condition.30 The steady state fluorescence spectra of DAPI−DNA with increasing amounts of [Mor1,2][Br] are shown in Figure 3. The observation of a decrease in

Table 2. Fluorescence Decay Parameters of DAPI−DNA (Lifetimes in ns) in the Presence of Different Quantities of [Mor1,2][Br] sample 0M 0.3 M 0.5 M 0.7 M 1.0 M

τ1 (a1) 1.40 1.30 1.35 1.31 1.16

(0.39) (0.35) (0.20) (0.18) (0.14)

τ2 (a2) 3.40 3.40 3.52 3.51 3.49

(0.61) (0.65) (0.38) (0.39) (0.33)

τ3 (a3)

0.16 (0.42) 0.17 (0.43) 0.25 (0.53)

3.4. Fluorescence Correlation Spectroscopy Study. FCS is a valuable tool to understand the conformational dynamics and to determine the size of biomolecules.33−36 FCS has been employed to investigate the DNA−protein interactions and to examine DNA hybridization.37,38 It is shown that FCS can be used to study the condensation of DNA using dyes which are noncovalently bound to DNA.39,40 Conformational dynamics of DNA has also been studied by attaching a dye molecule covalently.41,42 In the present study, FCS measurements have been performed to determine the hydrodynamic radius (Rh) of DNA in the presence of different quantities of [Mor1,2][Br] and also to substantiate its groove binding. For this purpose, we have monitored the fluorescence of DAPI−DNA, using a solution containing 150 nM DAPI and 50 μM DNA. DAPI is expected to be fully bound to DNA under this condition as well and exhibit a strong fluorescence. Even if few molecules are in the unbound state, they are not expected to contribute to the correlation data because of their weak emission in an aqueous environment. The fluorescence correlation data of DAPI−DNA is shown in Figure 5 along with fits to eq 3, which is based on simple diffusion, and eq 4, which includes simple diffusion along with intersystem crossing. It is clear from the fit residuals that eq 4 represents the data adequately and is, hence, used for further analysis.

Figure 3. Fluorescence spectra (λexc = 375 nm) of DNA-bound DAPI (DAPI−DNA, 3 μM DAPI and 60 μM DNA) in the presence of 0, 0.1, 0.3, 0.5, 0.7, and 1.0 M [Mor1,2][Br].

fluorescence intensity of the system with an increase in the concentration of IL indicates that [Mor1,2]+ expels the DAPI molecule from the minor grooves of DNA. The measured fluorescence decay profiles of DAPI−DNA as a function of the concentration of [Mor1,2][Br] are shown in Figure 4. The estimated decay parameters (Table 2) were obtained from biexponential fits, I(t) = a1 exp(−t/τ1) + a2 exp(−t/τ2), where τ1 and τ2 are individual lifetime components and a1 and a2 are associated amplitudes at t = 0. The fluorescence decay of DAPI−DNA is found to be biexponential with lifetimes of 1.4 and 3.4 ns. As the two lifetime values are typical of the two forms of DAPI in its DNA-bound state, it is evident that, in the absence of [Mor1,2][Br], DAPI is almost totally bound to DNA31,32 with negligible exposure to the aqueous environment. The appearance of a short-lived third component at higher concentrations of [Mor1,2][Br] is consistent with the release of DAPI into the aqueous environment from DNA. Thus, both docking and fluorescence studies clearly suggest that [Mor1,2]+ binds to the minor grooves of DNA.

G (τ ) =

G(τ ) =

−1/2 −1 1⎛ τ ⎞ ⎛ τ ⎞ ⎜1 + ⎟ ⎜1 + 2 ⎟ N⎝ τD ⎠ ⎝ κ τD ⎠

(3)

−1/2 −1 1 − T + T exp( − τ /τtr) ⎛ τ ⎞ ⎛ τ ⎞ ⎜1 + ⎟ ⎜1 + 2 ⎟ τD ⎠ ⎝ N (1 − T ) κ τD ⎠ ⎝

(4)

Here, τD is the diffusion time, N represents the number of molecules in the observation volume, and κ is the structure D


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Figure 5. Correlation data of DAPI−DNA in Tris−HCl buffer (pH 7.4) along with fits to a simple diffusion model, eq 3 (A), and simple diffusion with intersystem crossing, eq 4 (B).

parameter defined as κ = (ωz/ωxy) in which ωz and ωxy are the longitudinal and transverse radii of the observation volume, respectively. The hydrodynamic radius of DNA is estimated using the following equation, which corrects for the viscosity and refractive index mismatch22 R hDNA R hRh6G

=

τDDNA τDRh6G

(5)

The Rh value of ct-DNA is found to be 137 ± 10 nm, which matches with the literature value.43 Figure 6 plots the estimated

Figure 7. Variation of the G(0) value with the concentration of [Mor1,2][Br].

3.5. DNA Melting Study. The thermal stability of DNA is monitored by measuring the absorbance at 260 nm as a function of temperature. It is known that DNA shows hyperchromism when its two strands are separated during the melting process.24 The melting curves of DNA in the presence of various quantities of [Mor1,2][Br] are shown in Figure 8. The melting temperatures in the presence of 0, 0.3, and 1.0 M [Mor1,2][Br] are estimated to be 75 ± 0.4, 78 ± 0.6, and 82 ± 0.1 °C, respectively. These results, which indicate that DNA Figure 6. Hydrodynamic radius (Rh) of DNA as a function of the concentration of [Mor1,2][Br].

Rh values of DNA as a function of the concentration of [Mor1,2][Br]. The near constancy of the Rh values for different concentrations of the ILs indicates a negligible change in the structure of DNA. This observation is consistent with the results of the CD experiment. The change in the G(0) values at different concentrations of [Mor1,2][Br] is shown in Figure 7. An increase in the G(0) value on addition of [Mor1,2][Br] suggests a decrease in the value of N, indicating expulsion of the DAPI molecules from its DNA-bound state. This finding is in accordance with the results of the steady-state and timeresolved fluorescence experiments. Thus, FCS results unambiguously establish that [Mor1,2]+ binds to minor grooves of DNA and it replaces the DAPI molecules from the grooves.

Figure 8. Melting curves of DNA (30 μM) in buffer (squares) and 0.3 M (circles) and 1.0 M (triangles) [Mor1,2][Br]. E


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(2) Liu, H.; Liu, D. DNA Nanomachines and Their Functional Evolution. Chem. Commun. 2009, 19, 2625−2636. (3) Yamada, M.; Yokota, M.; Kaya, M.; Satoh, S.; Jonganurakkun, B.; Nomizu, M.; Nish, N. Preparation of Novel Bio-matrix by the Complexation of DNA and Metal Ions. Polymer 2005, 46, 10102− 10112. (4) Zhao, H. DNA Stability in Ionic Liquids and Deep Eutectic Solvents. J. Chem. Technol. Biotechnol. 2015, 90, 19−25. (5) Lukin, M.; Santos, C. D. L. NMR Structures of Damaged DNA. Chem. Rev. 2006, 106, 607−686. (6) Chandran, A.; Ghoshdastidar, D.; Senapati, S. Groove Binding Mechanism of Ionic Liquids: A Key Factor in Long-Term Stability of DNA in Hydrated Ionic Liquids? J. Am. Chem. Soc. 2012, 134, 20330− 20339. (7) Vijayaraghavan, R.; Izgorodin, A.; Ganesh, V.; Surianarayanan, M.; MacFarlane, D. R. Long-Term Structural and Chemical Stability of DNA in Hydrated Ionic Liquids. Angew. Chem., Int. Ed. 2010, 49, 1631−1633. (8) Bonner, G.; Klibanov, A. M. Structural Stability of DNA in Nonaqueous Solvents. Biotechnol. Bioeng. 2000, 68, 339−344. (9) Hammouda, B.; Worcester, D. The Denaturation Transition of DNA in Mixed Solvents. Biophys. J. 2006, 91, 2237−2242. (10) Park, S.; Kazlauskas, R. J. Biocatalysis in Ionic Liquids − Advantages Beyond Green Technology. Curr. Opin. Biotechnol. 2003, 14, 432−437. (11) Wei, W.; Danielson, N. D. Fluorescence and Circular Dichroism Spectroscopy of Cytochrome c in Alkylammonium formate Ionic Liquids. Biomacromolecules 2011, 112, 2566−2572. (12) Nishimura, N.; Nomura, Y.; Nakamura, N.; Ohno, H. DNA Strands Robed with Ionic Liquid Moiety. Biomaterials 2005, 26, 5558− 5563. (13) Qin, W.; Li, S. F. Y. Electrophoresis of DNA in Ionic Liquid Coated Capillary. Analyst 2003, 128, 37−41. (14) Wang, J. H.; Cheng, D. H.; Chen, X. W.; Du, Z.; Fang, Z. L. Direct Extraction of Double-Stranded DNA Into Ionic Liquid 1-Butyl3-methylimidazolium Hexafluorophosphate and Its Quantification. Anal. Chem. 2007, 79, 620−625. (15) Ding, Y.; Zhang, L.; Xie, J.; Guo, R. Binding Characteristics and Molecular Mechanism of Interaction between Ionic Liquid and DNA. J. Phys. Chem. B 2010, 114, 2033−2043. (16) Xie, Y. N.; Wang, S. F.; Zhang, Z. L.; Pang, D. W. Interaction Between Room Temperature Ionic Liquid [bmim]BF4 and DNA Investigated by Electrochemical Micromethod. J. Phys. Chem. B 2008, 112, 9864−9868. (17) Wang, H.; Wang, J.; Zhang, S. Binding Gibbs Energy of Ionic Liquids to Calf Thymus DNA: A Fluorescence Spectroscopy Study. Phys. Chem. Chem. Phys. 2011, 13, 3906−3910. (18) Cheng, D. H.; Chen, X. W.; Wang, J. H.; Fang, Z. L. An Abnormal Resonance Light Scattering Arising from Ionic-liquid/ DNA/ethidium Interactions. Chem.Eur. J. 2007, 13, 4833−4839. (19) Jumbri, K.; Rahman, M. B. A.; Abdulmalek, E.; Ahmadab, H.; Micaelo, N. M. An Insight into Structure and Stability of DNA in Ionic Liquids from Molecular Dynamics Simulation and Experimental Studies. Phys. Chem. Chem. Phys. 2014, 16, 14036−14046. (20) The melting point of [Mor1,2][Br] is 190 °C. Strictly speaking, it is not an ionic liquid. However, as choline dihydrogen phosphate (mp 195 °C) is described as an ionic liquid, we follow the same trend. (21) Petkovic, M.; Seddon, K. R.; Rebelo, L. P. N.; Pereira, C. S. Ionic Liquids: A Pathway to Environmental Acceptability. Chem. Soc. Rev. 2011, 40, 1383−1403. (22) Pabbathi, A.; Ghosh, S.; Samanta, A. FCS Study of the Structural Stability of Lysozyme in the Presence of Morpholinium Salts. J. Phys. Chem. B 2013, 117, 16587−16593. (23) Patra, S.; Santhosh, K.; Pabbathi, A.; Samanta, A. Diffusion of Organic Dyes in Bovine Serum Albumin Solution Studied by Fluorescence Correlation Spectroscopy. RSC Adv. 2012, 2, 6079− 6086. (24) Mati, S. S.; Roy, S. S.; Chall, S.; Bhattacharya, S.; Bhattacharya, S. C. Unveiling the Groove Binding Mechanism of a Biocompatible

stability is enhanced in the presence of IL, are in line with the earlier observations of enhanced thermal stability of DNA in choline and imidazole based ionic liquids.7,19 The observation, i.e., an enhanced thermal stability of DNA in the presence of the IL, can be explained following the mechanism suggested by Auffinger and Jumbri.19,44 According to them, the hydration shell around DNA is responsible for its thermal stability and with an increase in temperature an enhanced entropy of water molecules destabilizes the DNA. However, in the presence of IL, the DNA is not only surrounded by water but also by the constituents of the IL. As the latter interacts with DNA, the entropy gain at higher temperature under this condition may not be enough to destabilize the DNA due to its strong interaction with the IL.

4.0. CONCLUSIONS Realizing the potential of the ILs in biological applications and noting that only a few studies involving the ILs and DNA have been carried out so far, we have investigated the structural integrity of DNA in the presence of a less toxic morpholinium IL, [Mor1,2][Br], and attempted to understand the nature of interaction between these two important classes of chemical substances. The results indicate that DNA retains its B form in the presence of [Mor1,2][Br]. It is shown that morpholinium cation binds to the minor grooves of DNA and its binding is slightly weaker compared to the imidazolium cation presumably due to a reduced number of interactions between morpholinium cation and DNA and nonplanarity of the present cation. The FCS study on DAPI-bound DNA not only substantiates groove binding of [Mor1,2]+, but it also reveals no significant change of the hydrodynamic radius of DNA in the presence of the IL, thereby confirming the structural integrity of DNA. Considering the low toxicity and greater biocompatibility of the morpholinium ILs compared to the imidazolium ones, the present results seem to brighten the prospect of practical utilization of these ILs in DNA technology.

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ASSOCIATED CONTENT

S Supporting Information *

CD spectra of DNA in the presence of IL at different incubation times. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.jpcb.5b02939.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the J.C. Bose Fellowship (to A.S.) and PURSE grant (to the University of Hyderabad) of the Department of Science and Technology, Government of India. We thank Dr. Karunakar Tanneeru of our school for helping us in the molecular docking study. A.P. thanks Council of Scientific and Industrial Research for the fellowship.

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REFERENCES

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