Influence of Ionic Liquids on Thermodynamics of Small MoleculeâDNA

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Influence of Ionic Liquids on Thermodynamics of Small MoleculeDNA Interaction: the Binding of Ethidium Bromide to Calf Thymus DNA Arpit Suresh Mishra, Mary Krishna Ekka, and Souvik Maiti J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11823 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016

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Influence Of Ionic Liquids On Thermodynamics Of Small Molecule-DNA Interaction: The Binding Of Ethidium Bromide To Calf Thymus DNA Arpit Mishra1#, Mary Krishna Ekka1#, Souvik Maiti1,2,3∗ 1

CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi 110 007, India.

2

Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India. 3

CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India. #

both the authors contributed equally.

*

To whom correspondence should be addressed: Tel: +91 11 27666156; Fax: +91 11 27667437; E-mail: [email protected]

ABSTRACT Ionic liquids (ILs) are salts with poor ionic co-ordination, resultantly remaining in liquid state below 100°C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-Ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ∆G of ctDNA-EB interaction is attributed to decreased entropy in case of ILs, while the same effect by NaCl was due to increased enthalpy.

INTRODUCTION Ionic liquids (ILs) are fast emerging as next generation solvents due to their unique physicochemical properties like near zero vapor pressure, low flammability, high ionic conductivity, good thermal conductivity, and increased electrochemical potential window.1 Ionic compounds consist of cations and anions held by electrostatic interaction in a regular lattice structure. Since these lattice structures have very high lattice enthalpies, ionic compounds have high melting temperature, for example, Sodium Chloride (NaCl) melts at temperature above 800°C. In the case of Ionic liquids, the cations usually are organic moieties of large size which decrease the lattice enthalpy due to increased conformational flexibility.2,3 It is due to this property of ILs that they characteristically remain in liquid state at temperatures below 100°C. ILs can further be categorized into room temperature ionic liquids (RTILs) if they remain in liquid form even at room temperature. The first major breakthrough in this field came from Gabriel who had reported ethanolammonium nitrate as an Ionic liquid in 1888.4 This was followed by Walden who reported ethylammonium nitrate in 1914, but due to high reactivity of this solvent it failed to capture the interest of the scientific community.4 Later on, in 1951, the first room temperature stable Ionic liquid N-ethylpyridinium bromide-aluminium chloride was discovered. With the discovery of 1,3-dialkylimidazolium salts in 1982 and 1-ethyl-3methylimidazolium tetrafluoroborate in 1992 novel avenues of water, air stable and hydrophobic/philic Ionic liquids opened up.5,6 ILs

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provide an environment friendly chemical alternative for many chemistry based applications like synthesis and catalysis, enzymatic immobilization, solvents for organic reaction, and electrochemical applications.4,5 Moreover, ILs are also known as “tailormade” solvents as their hydrophilic/hydrophobic properties can be varied by replacing the anions. 7 Due to adjustable hydrophilic/phobic properties, ILs provide a unique microenvironment for many biological applications including but not limited to, enzymatic reaction, biotransformation , gene delivery and for long term stability of nucleic acids and proteins.8 It has been shown that lipase from Candida antarctica shows higher stability in longer hydrophobic alkyl chain containing ILs.9 Pretreatment of cellulose with ILs also aids cellulase activity.10 ILs are also known to provide an excellent IL/water based biphasic system for Lactobacillus kefir whole cell based asymmetric reduction reaction.11 ILs are also used for pretreatment of lignocellulosic material to remove lignin so microbes can utilize cellulose for biofuel production.12 In 1997, novel pyridinium surfactants were used for efficient gene delivery.13 Recently Chen et al used imidazolium based polymeric ionic liquids for successful gene delivery.14 Together, these and many other evidences indicate that ILs are amicable to biological systems constituted by biological macromolecules like protein and DNA. Recently, a new category of ILs, known as Bio-ILs, made entirely from biomaterials like choline acetate, choline dihydrogen phosphate (dhp) and choline lactate has been reported.15 Biophysical interaction of these ILs with DNA and siRNA is well explored along with their effect on stabilization and nuclease protection of these nucleic acids.15,16,17 Since DNA is an excellent chiral substrate for enzymatic reaction and DNA structures are acquiring popularity in nanotechnology based applications, DNA structural stability has been studied in many novel IL based solvents.18 Binding of DNA to 1- butyl- 3-methylimidazolium chloride (BMIM-Cl) has been explored, where Ding et al have shown that BMIM-Cl interacts with DNA through its cationic headgroup and compacts DNA.19 Similar report for another ionic liquid guanidinium tris(pentafluoroethyl) trifluorophosphate (Gua-IL) shows cationic headgroup interaction of ILs with the negatively charged phosphate backbone of DNA that helps in compaction of DNA structure.20 Although only a few studies have explored effect of ILs on DNA stability, none of the studies have explored thermodynamic effects of Ionic liquid based solvents on DNA-small molecule interaction. Synthetic small molecules are coming up rapidly as novel therapeutic targets as they can modify gene expression via interaction with specific DNA sequence.21 In chemical biology many small molecule screens are used for identifying novel drug candidates.22 Small molecule solubility varies in different solvents and Ionic liquids can provide an alternative choice of solvents due to enhanced solvent power.23,24 Traditionally small molecules have been used as visualizing tools in the form of DNA staining dyes.25,26 Alternatively, their effects on replication, transcription or DNA modifying abilities have also been studied25,27 These molecules generally find application in drug therapy for cancer and hence are therapeutically relevant.25,26 Some DNA-binding ligands can act as probes for DNA damage.28 Small molecules interact with DNA by two major modes of action; by binding to the minor groove, and by intercalating between base pairs. 29,30 Because these molecules are of general importance and are involved in regulation of cellular machinery, understanding the interactions between DNA-small molecules are of utmost importance. ILs are potential alternative solvents for DNA binding molecular events like DNA-protein interaction, DNA-RNA interaction or DNA-small molecule interaction. In this context, it is very important to understand the thermodynamic parameters for these interactions in presence of novel hydrated IL based solvents. Herein, we have tried to explore the effect of different types of hydrated ILs at different concentrations on DNA-small molecule interaction. We have used ctDNA-EtBr (ctDNA-EB) interaction as a model for DNA-small molecule interaction where we have explored the effect of ILs and NaCl on DNA stability using UV melting experiments. ctDNA-EB interaction has been well studied using UV absorption spectroscopy, fluorescence quenching, molecular docking studies and hence is a good model system for monitoring IL based changes. EB binds to ctDNA by intercalating DNA through interactions with the minor groove .31,32 IL- mediated minor perturbations on DNA were explored using CD spectroscopy and


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steady state fluorescence mediated EB displacement assays. Moreover, we also compared the effect of Ionic liquids with commonly used ionic solution - NaCl. Sodium ions have been shown to stabilize nucleic acid structures, mainly helices. Increasing salt concentrations lead to a corresponding increase in the number of ions bound to DNA, thus resulting in a lower electrostatic free energy. In the unbound state, the ion has larger entropy because it can move around in the bulk solvent. After binding to DNA, the ion is mostly fixed and thus the bound entropy is smaller. The ions bound to the phosphate backbone of dsDNA lead to reduction of repulsive Coulombic interactions, thus leading to higher helix stability.33 Thermodynamic parameters for ctDNA-EB interactions in the presence of different types of ILs and NaCl at a particular concentration have also been studied. We also calculated entropic and enthalpic contributions for all ILs and NaCl on ctDNA-EB interaction using Isothermal calorimetric based experiments. Comparisons of thermodynamic parameters of ILs with NaCl solution indicate a contrast between their thermodynamic signatures. Our results also suggest that effect of ILs and NaCl on ∆G is similar, but contributing factors for this effect are different.

MATERIALS AND METHODS Sodium salt of ctDNA (Calf Thymus DNA) and EB (3, 8- diamino-5-ethyl-6-phenylphenanthridium bromide), 1- butyl- 3methylimidazolium chloride (BMIM-Cl), 1- butyl- 3-methylimidazolium bromide (BMIM-Br), 2-Hydroxyethyl-trimethylammonium(+)-lactate choline lactate or (HETAL) and choline acetate were purchased from Sigma-Aldrich and used for all experiments. ctDNA and EB were dissolved in 10 mM Sodium Phosphate buffer (pH7.0) at room temperature and left overnight on a rotator. After complete dissolution, concentration of DNA and EB solution was estimated using Varian UV-Vis spectrophotometer. Molar extinction coefficient (ε = 13200 M-1cm-1) and absorbance at 260 nm was used to calculate concentration of ctDNA in base pairs, for Ethidium bromide, molar extinction coefficient (ε = 5600 M-1cm-1) and absorbance at 480 nm was used. ctDNA and EB stock solutions were stored at 4°C until further use. All DNA estimations were taken in base pairs.

Circular Dichroism CD spectra were recorded to detect structural alterations in ctDNA using a Jasco 815 CD spectrophotometer. Briefly, 58 µM ctDNA was incubated with different concentrations of ILs and NaCl (100 mM to 500 mM) for 5 minutes and CD spectra was collected. The spectra obtained were an average of three consecutive scans for each sample with a path length of 10 mm and a bandwidth of 1 nm.

UV melting curves of ctDNA in presence of different Ionic liquids. UV melting experiments were performed using a Cary 100 (Varian) spectrophotometer equipped with a thermoelectrically controlled cell holder. ctDNA was dissolved in 10 mM sodium phosphate buffer (pH7.0) to a final concentration of 58 µM. Increasing concentration (100 mM to 500 mM) of different ILs was added and mixed thoroughly. Melting curve profiles of these different ratios of IL to DNA were obtained by collecting absorbance of IL-DNA complexes at 260 nm as a function of temperature. A temperature range of 40−100 °C was used to monitor the changes in absorbance at 260 nm at a heating rate of 0.5 °C/min. The melting curves obtained were plotted using Origin 7.0 and Tm was calculated by taking median of higher baseline and lower base line drawn on melting curve as described by Mergny and Lacroix.34

Steady-State Fluorescence Spectroscopic Studies For estimating DNA compaction effect of different ILs, EB Exclusion assay was used. In EB exclusion assay, steady state fluorescence spectra were collected in Jobin Yvon Fluorolog spectrofluorometer. The excitation wavelength for EB was 480 nm and emission spectra were collected from 500 to 700 nm range using a slit width of 5 nm. All spectra were collected at 25 °C. EB-DNA



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complex was prepared by mixing 90 µM EB with 180 µM DNA in 10 mM Sodium phosphate buffer in a 500 µl reaction in a quartz cuvette. Increasing concentration of different ILs and NaCl (from 100 mM to 500 mM) was added and fluorescence spectra (I) were collected. Data was normalized using the fluorescence intensity of only DNA in buffer (I0). For measuring the EB exclusion capacity for each IL and NaCl, intensities [I] and [I0] were taken at 600 nm. Data was plotted using Origin 7.0. To confirm that IL does not affect EB fluorescence, control experiments in presence of only IL and EB were performed. While there was no change on EB fluorescence due to Choline based ILs, minor increase in fluorescence was observed with Imidazolium based IL (Supporting Information Fig S1).

Isothermal Titration Microcalorimetry Isothermal titration calorimetry (ITC) experiments were carried out to understand the effect of varied ILs on DNA-small molecule interaction using Microcal VP-ITC (Microcal, INC;Northampton MA). ctDNA concentration was kept constant at 30 µM and EB concentration was 400 µM. DNA and small molecule (EB) were dissolved in 10 mM sodium phosphate buffer and equal concentration (300 mM) of ILs were added to both solutions. Titrations of DNA and EB in presence of ILs were carried out at 25°C. Along with this titration similar experiments were performed without ILs and in presence of NaCl. The duration of each injection was 20 seconds and delays between injections were 180 seconds with stirring. All injection volumes were 10 µl except first, which was 2 µl. Each injection produced a heat burst curve (microcalories/second vs. second) and area under the curve was integrated using Microcal ITC analysis software. The ITC profiles for the binding of ctDNA-EB in different ILs was fit with one binding site model. For ITC experiments done in 1M IL solution, the thermograms obtained were extremely noisy. A probable explanation could be because of the higher viscosity of the solution. Thus, ITC was not successful at this IL concentration. Lower concentrations (< 0.5 M) were sufficient to induce an effect on ctDNA-EB interaction and hence, higher concentrations of ILs (>0.3 M) were not used for ITC experiments.

RESULTS In this study we attempted to understand the effect of different ILs and their concentrations on DNA- small molecule interactions. Here we used four different hydrated Ionic liquids, majorly divided into two classes- Imidazolium based ionic liquids (BMIM-Cl and BMIM-Br) and choline based Ionic liquids (choline acetate and choline lactate). Both classes of ILs are highly hydrophilic.16,19 Imidazolium based ILs have Chlorine and Bromine as anions and 3-methylimidazolium ring as cation, while in choline based ILs choline acts as cation and is paired with lactate and acetate based anions as shown in Scheme 1.

ILs do not induce major conformational changes in DNA CD spectroscopy was performed to detect any structural alterations in ctDNA caused due to the presence of employed ILs at different concentrations. CD spectra of pure ctDNA in 10 mM sodium phosphate buffer and the spectra of ctDNA in presence of a concentration gradient of different ILs (100 mM to 500 mM) were collected using a Jasco 815 spectrophotometer. CD spectrum of ctDNA in absence of ILs or NaCl shows two major peaks: one positive maxima at 278 nm corresponding to π-π base packing and a negative maxima around 240 nm corresponding to DNA helicity (Figure 1 panel a-e). Both positive and negative peaks are of equal amplitude; a characteristic signature of B-form of DNA.35 Even after increasing the concentration of ILs up to 500 mM, no major difference in CD signature was observed (Figure 1 panel b-e). Although intensity of positive peaks around 275 nm is a bit reduced (Figure 1, panel f) indicating some disturbance in DNA structure, overall ctDNA retained its native B-form as reported previously. 16,19

IL-induced structural changes were similar to NaCl-induced changes at comparable concentration, further suggesting that ILs do


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not cause significant alterations in ctDNA structure (Figure 1, panel a-e). These minor structural changes in DNA via ILs can be attributed to the interaction of cationic group of IL with the negatively charged phosphate backbone of DNA. However, more detailed studies would be required to understand this observation.

Thermal melting suggests that ILs increase DNA stability To understand the effect of various ILs on DNA stability, we performed thermal melting experiments of ctDNA in presence of ILs and NaCl. UV based thermal melting displayed a characteristic hyperchromic shift (increase in absorbance with increasing temperature) indicating the denaturation of double stranded ctDNA to single stranded form. The melting curves obtained (Figure 2, panel a-c) were further analyzed to determine the thermal stability of ctDNA in presence of different ILs and NaCl. The melting temperature (Tm) of ctDNA in buffer was 69°C which increased to 90°C in presence of NaCl (Figure 2, panel e). Tm value obtained in presence of Imidazolium based ILs was 74°C and in presence of choline based ILs was 89°C suggesting a stabilizing effect of ILs on ctDNA. This variable hyperchromicity in case of ILs can be due to the different extents of polarity induced by the ILs in the bulk solvent. Moreover the increase in DNA stability is more pronounced for choline based ILs than Imidazolium based ILs. The effect of increased stability was concentration dependent and on par with NaCl further highlighting that ILs can increase DNA stability, but the stabilization effect varies with different group of ILs. Interestingly IL- mediated stabilization does not affect DNA structure, thus maintaining its native B-form.

EB is excluded from DNA in presence of ILs CD spectra indicated that DNA retains predominantly B-form showing minor structural changes with increasing concentration of ILs. To explore if these structural changes interfere with the capability of DNA to interact with small molecules, we tested ILs’ effect on ctDNA-EB interaction using EB exclusion assay. Steady state fluorescence spectroscopy was used to gain insight into DNA-small molecule (ctDNA-EB) interaction. Fluorescence intensity of unbound or free EB is less than DNA bound EB because, in bound form EB is in a hydrophobic microenvironment, wherein fluorescent quenchers like water and oxygen cannot access EB as freely as in the unbound form.36 Fluorescence intensity (a.u) was measured and a characteristic EB exclusion spectra was plotted for ctDNA-EB complex without any salt or ILs. Subsequently for all four ILs, a concentration range of 100 mM IL to 500 mM was added to the ctDNA-EB complex and fluorescence spectra (I) were collected (Figure 3, panel a-e). For each IL concentration, normalized fluorescent intensity ([I]/[I0]) was plotted against concentrations of ILs (Figure 3, panel f). Literature suggests some of the ILs like BMIM-Cl and Gua-IL are known to compact DNA.19,20 Cationic groups of the ILs electrostatically interact with the charged phosphate backbone of DNA. This results in DNA compaction due to which there is a space constraint for EB to intercalate and intercalated EB excludes out. Hence we observe reduced fluorescence intensity with increasing IL concentration. Our results suggest different degrees of EB exclusion for imidazolium and choline based ILs. Imidazolium ILs showed around 40% reduction in fluorescent intensity while choline based ILs and NaCl showed fluorescence reduction of around 25 to 35%. These observations indicate that imidazolium ring containing ILs exclude more EB than choline ILs or NaCl.

Effect of different ILs on thermodynamics of DNA small molecule interaction To understand the effect of ILs on ctDNA-EB binding thermodynamics we performed ITC experiments (see Methods). ITC experiments for the two groups of ILs and NaCl were performed at concentrations of 300 mM ILs/NaCl. We calculated the thermodynamic parameters by titrating EB against ctDNA in presence of 300 mM ILs and NaCl. Appropriate concentration of ctDNA was maintained in the ITC cell and titrated with gradual addition of EB from the syringe. Titration was continued until EB



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concentration reached 10 times in excess of ctDNA in the cell. The integrated heat data of ctDNA-EB in blank and in presence of four different ILs and NaCl is shown in Figure 4. Calculated thermodynamics parameters from each titration are listed in Table 1. For all the titrations, integrated data was fitted with single binding site model with acceptable χ2 value. One-site binding model was used for fitting because binding of EB to DNA is well established; one EB molecule binds per two basepairs of DNA. Moreover, all binding sites of EB are similar in nature. Also, it has been suggested that effect of cooperativity does not function at salt concentrations beyond 50 mM.31 We performed ITC experiments at 300 mM IL concentrations and thus, single site binding model was appropriate and it gave good fitting results. Stoichiometry of binding was found to be close to 0.5 which is in accordance with reported binding stoichiometry of ctDNA-EB in buffer alone condition.21 Reduced stoichiometries of binding were observed in the presence of ILs and NaCl. Stoichiometries for binding in buffer condition and NaCl are 0.49 and 0.44 respectively. Similarly stoichiometries are 0.40 in BMIM-Cl and 0.41 in BMIM-Br. whereas it is 0.36 in choline acetate and 0.35 in choline lactate buffer. However, binding affinities Kbs reduced to almost half in presence of ILs or NaCl (Table 1) when compared with the binding affinity that is obtained in buffer alone condition. Binding affinities Kb in buffer alone condition is 5.79 × 105 mol-1. EB binding affinity in presence of salt is reduced to 2.14 × 105 mol-1. Kbs are 2.45 × 105 mol-1 and 2.11× 105 mol-1 in presence of BMIM-Cl and BMIM-Br respectively, while Kbs are 1.99 × 105 mol-1 and 1.92 × 105 mol-1 in the presence of choline acetate and choline lactate respectively.

EB binding to ctDNA in absence of ILs or NaCl has favorable ∆G, (-7.8 kcal mol-1) with majorly favorable enthalpic contribution ∆H -9.3 kcal mol-1 and unfavorable entropic contribution T∆S is -1.4 kcal mol-1. In presence of NaCl or ILs, ∆G remains favorable (around -7.2 kcal mol-1) with minor reduction in values. However, enthalpic and entropic contributions to binding energies in presence of NaCl are quite different, that is binding energy is contributed by lower amount of favorable ∆H (-6.3 kcal mol-1) and a small amount of favorable T∆S (0.9 kcal mol-1). EB binding energies in presence of both categories of ILs are more or less similar. In Imidazolium ILs, which consist of BMIM-Cl and BMIM-Br , ∆H for both cases are -8.73 and -8.09 kcal mol-1 which is lower than NaCl by -2.33 and 1.79 kcal mol-1 respectively but approximately 0.6 and 1.2 kcal mol-1 higher than the values that are obtained under buffer condition. Values of entropic contributions (T∆S) are -1.4 and -0.8 kcal mol-1 for BMIM-Cl and BMIM-Br respectively which are almost similar to the values obtained under buffer alone condition. We obtained favorable ∆H values of -8.9 and -9.6 kcal mol-1 for binding in presence of choline acetate and choline lactate ILs. Enthalpically choline based ILs is more similar to controlled buffer condition than that of Imidazolium ILs. T∆S values for choline acetate and choline lactate are -1.8 and -2.4 kcal mol-1, which is approximately 0.4 to 0.9 kcal mol-1 lower than controlled buffer condition that indicates in presence of choline based ILs system become more structured. This could be attributed to the mechanism of choline which binds to minor groove thus displacing water from the spine of hydration. When EB binds to ctDNA in choline containing buffer, it makes the DNA-IL-EB complex more hydrophobic which is compensated by formation of structured water around DNA-IL-EB complex thus decreasing entropy. As reported by Ding et. al., when the BMIM concentration is higher (>60 mM in case of BMIM Cl), the cationic headgroups of the ILs are placed near DNA phosphates, and the hydrocarbon chains are perpendicularly attached to the DNA surface leading to hydrophobic interaction between the hydrocarbon chains of BMIM and the DNA bases.19 In case of BMIM containing buffer, ctDNA-IL complex is already hydrophobic in nature, and upon binding to EB, DNA-IL-EB does not undergo any such compensation by formation of structured water around DNA-IL-EB complex and thus shows lower negative entropy change.

Comparisons of differential entropy ∆T∆S and differential enthalpy ∆∆H


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Differential thermodynamic parameters are calculated to obtain insight into the differential effect of salt and ILs, where thermodynamic parameters of DNA-EB interaction in buffer condition are subtracted from the thermodynamic parameters that are obtained under ILs and NaCl buffer conditions (∆∆G, ∆∆H and ∆T∆S). There parameters are presented in Table 1. Although the ILs exhibited different effects in terms of binding of EB to ctDNA, these effects were small and depict that EB-ctDNA interaction has similar thermodynamic signature (see figure 5) in the presence of NaCl and Imidazolium based ionic liquids but has different signature in presence of choline based ionic liquids. Positive values of ∆∆G indicate that binding becomes energetically unfavorable, positive values of ∆∆H indicate decrease in enthalpic contribution to the binding energies whereas positive values of ∆T∆S indicate increase in entropic contribution to the binding energies. All these three parameters are positive in case of EB-ctDNA interaction in presence of NaCl and Imidazolium based ionic liquids. In the case of choline lactate buffer, only ∆T∆S is having positive values and in the case of choline acetate buffer ∆∆G and ∆T∆S have positive values. This also indicates that the thermodynamic signature of EBctDNA binding for the two groups of IL studied here are different.

DISCUSSION In this study, four hydrated ionic liquids are analyzed for their effect on ctDNA structure stability and ctDNA-EB interaction. These four ILs which belong to either imidazolium or choline based class are explored for their effect on ctDNA-EB interaction, using CD spectroscopy, UV melting experiments and EB exclusion assay. ILs’ putative role as a novel solvent for DNA-small molecule interaction and their effect on thermodynamics of binding were explored using isothermal titration calorimetry. Even though CD spectral analysis (Figure 1) indicated that B-form of DNA structure is primarily retained in NaCl as well as in all the four Ionic liquids, there were some minor structural perturbations at the 275 nm positive peaks, in response to increasing concentration. The observed perturbation in DNA structure could be due to dehydration in presence of ILs. The hydration of the nucleic acids controls their structure and mechanism of action. Hydration of nucleic acids is attributed to the formation of a hydrogenbonded network of water surrounding the nucleic acid surface.37 The formation of this water network is highly sensitive to the water activity of the solution.38 B-DNA possessing higher phosphate hydration, less exposed sugar residues and smaller hydrophobic surface is stabilized at high water activity. It is known that either salt or ILs lower the water activity and hence, it can be argued that the structural perturbation observed either in presence of NaCl or ILs is due to reduction of water activities. Slightly higher structural perturbation noticed in presence of ILs than in the presence of NaCl (Figure 1, panel f) is due to the fact that water activities in presence of ILs are slightly lower than the water activity in presence of NaCl.38 The UV melting experiments (Figure 2) suggest that both the classes of ILs behave differently although both lead to an increase in the stability of DNA helix, evident by corresponding Tm values. Choline based ILs stabilize ctDNA more than Imidazolium based ILs. Moreover, Imidazolium based ILs’ stabilization effect gets saturated at 100 mM concentration but Choline based ILs’ stabilization effect increases with increase in the concentration of ILs. Similar stabilization effect saturation from Imidazolium based IL was also observed in recent reports from Khimji et al, where they demonstrated that partially soluble IL BMIM-PF6 shows minor increase (1°C) in Tm. Water miscible BMIM-BF4 shows increase in Tm from 37 °C to 43 °C at 100 mM but increasing the concentration further showed an opposite effect on Tm.39 These differences in stabilization effects of Choline and Imidazolium based ILs can be explained using their DNA binding mechanism. ILs as well as NaCl stabilizes DNA due to electrostatic interaction between their respective cation and phosphate backbone. This interaction causes removal of the hydration shell around the phosphate backbone. In case of Choline, it has been shown that Choline ions can interact with phosphate backbone as well as with ribose sugars and nucleobases



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through hydrogen bond formation between the hydroxyl group of choline ion and DNA.40 Choline ions bind three times more DNA over Na+ ion from NaCl, due to their multiple hydrogen bonding capability.40 Moreover, Choline has been shown to preferentially bind at minor groove at AT rich site.41 At higher concentration Choline ions are also shown to bind to the major groove and form two hydrogen bonds. Moreover, Choline shows preferential stabilization of AT rich dsDNA.42 The ctDNA used in this study is 58% AT rich. This explains the increased DNA stability in presence of choline when compared to imidazolium ring based ILs. Previous literature shows that up to 50% (w/w) solution of imidazolium ring based ILs majorly interact with phosphate backbone of DNA.43 Hence, only one mode of interaction takes place in this case unlike choline where multimodal binding takes place. Likewise, stabilization effect is not as pronounced as seen in the case of choline. This comparison indicates that Bio-ILs which are choline based have more DNA stabilization potential when compared to traditional imidazolium ring based ILs. Differential dependence of the Tm on IL and Na+ concentration In previous reports, concentration of ILs used for stability studies are very high (4M) in comparison to our study (500 mM). Variations in binding affinities and effect on Tm could exist depending on the concentration of IL used, which is recently reported in case of BMIM-BF4, where till 100mM it shows Tm stabilization but further increase in concentration leads to reversible effect on Tm.39 Although our study does not probe the binding mechanism of ILs, but probable reasons for BMIM saturation at 100mM could be that at lower concentration (upto 50% w/w) BMIM is known to interact majorly with DNA phosphate backbone.43 It is surprising that choline ILs show higher binding and Tm stabilization than Imidazolium ILs, which is in contradiction to previous report from Chandran et al.37 Choline shows preferential interaction with minor groove or AT and is also shown to bind at major groove and phosphate backbone although with less affinity than the minor groove.40,42 Due to this it is possible that BMIM has limited binding sites available and thus gets saturated early while choline has more binding sites available and thus does not get saturated. It has also been shown that cholines’ binding is three times stronger than NaCl.40,41 It is quite surprising that this strong binding is not reflected in equivalent Tm increase when compared with NaCl. This could be explained with cholines’ unique binding preference where it stabilizes AT- rich DNA via minor groove binding but destabilizes GC- rich dsDNA.42 Thus, Cholines’ strong binding effect may get nullified because of its destabilizing effect on GC- rich dsDNA and hence is not reflected with a corresponding increase in Tm . In EB displacement assay, fluorescence intensity was reduced upon increasing concentration of ILs which indicates EB is excluded out of intercalated state. Hence, EB’s fluorescence gets quenched due to absence of hydrophobic environment provided by DNA intercalation. When EB exclusion capacities were compared with ILs and NaCl, Imidazolium ring based ILs show up to 35 to 40% reduction in EB fluorescence while Choline based ILs and NaCl based solvents show 25-30 % reduction. Increased EB exclusion for imidazolium based ILs can be explained by previous MD simulation based studies where Micaelo group has shown that in presence of hydrated imidazolium IL solution upto 50% (w/w), imidazolium cations bind to the phosphate backbone perpendicular to the DNA helix. The binding of the cation to the phosphate backbone displaces water from phosphate and water diffuses inside the helical structure which disturbs the amine stacking thus quenching EB fluorescence.43 Contradiction between UV melting and EB exclusion studies Cations of ILs bind to phosphate to shield the negative charge on the phosphate of natural DNA through electrostatic interactions. However, molecular ions like choline and Imidazolium bind to DNA through other varied modes like hydrogen bonding, and van der Waals interactions in addition to electrostatic interaction because of their sizes and chemical characteristics. It is reported in the literature that choline ion interacts with DNA by forming hydrogen bonds with many DNA atoms in addition to electrostatic interactions.40 Such additional hydrogen bond formation is absent in case of Imidazolium binding to DNA. The higher stabilities of DNA observed in case of choline based ILs could be due to this additional stabilization that happens through hydrogen bond


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formation. In case of EB exclusion experiments, as the cationic group of ILs bind to DNA through electrostatic interaction in addition to other interactions, it weakens the electrostatic interaction between DNA phosphates and EB cations leading to gradual release of EB from DNA to the bulk water and, thus, the decrease in the fluorescence intensity of EB. As discussed previously, observation from Ding et. al. indicates that when the BMIM concentration is above 60 mM the cationic headgroups of the ILs are placed near DNA phosphates, and the hydrocarbon chains are perpendicularly attached to the DNA surface leading to hydrophobic interaction between the hydrocarbon chains of BMIM and the bases of DNA.19 Such interaction is not possible when choline based ILs interact with DNA. These additional hydrophobic interactions lead to more EB exclusion in case of BMIM ILs than Choline based ILs. Comparison of IL and NaCl induced changes on EB-ctDNA interaction Na+ binds mainly through electrostatic interaction with phosphate backbone while IL binding is multimodal and they also replace water from minor groove, major groove and from the spine of hydration. So DNA-IL complex is more hydrophobic which is rehydrated through formation of structured water cages which lead to more unfavorable entropy than NaCl. In the case of NaCl binding, Na+ binds to phosphate backbone of DNA thus replacing the structured water cage around phosphate which leads to reduction of structured components and thus NaCl binding is entropically driven. Na+ binding does not remove water from minor groove thus overall hydrophobic-hydrophilic balance is not disturbed to DNA-IL bindings’ extent. Moreover, in ITC we have pre-incubated IL and NaCl along with DNA, thus most of the EB binding sites are preoccupied. Due to these preoccupied binding sites, binding affinity for EB is reduced to almost half. EB binding is governed by EB induced change in DNA structure but due to presence of NaCl or IL this change in structure is resisted to some extent by reducing binding affinities for EB.

Entropic-Enthalpic compensation in ctDNA-EB interaction Upon comparison of our result with existing literature, we found the basis for different enthalpic changes for different ILs, as suggested by a study from Marky in which they compared differences in the intercalation and thermodynamics of structurally similar phenanthroline intercalators like PI(Propidium Iodide) and EB.31 Marky et al have suggested that difference in ∆H of binding depends primarily due to varied levels of hydration by different intercalators.31 Moreover, they also found that depending on sequence and its hydration status, reaction is either driven by entropic or enthalpic changes for dye intercalation. In their paper they also confirmed that change in entropy ∆S is directly proportional to volume change ∆V°. Thermodynamic basis for entropic contribution by NaCl and enthalpic contribution by ILs can be similarly governed by difference in ∆V°, due to different level of dehydration upon NaCl and IL binding. Despite different governing contribution on DNA-EB interaction in presence of NaCl or ILs, feasibility of reaction or ∆G is almost identical in both cases. This is a classic entropy-enthalpic compensation phenomenon. Chou et al discussed this concept using PI and EB binding to many different DNA substrates.44 Binding of EB or PI leads to the release of counter-ions which contribute to binding entropy, binding induced dehydration also contributes to binding entropy and the same molecular processes could also lead to reducing binding enthalpy. These kind of molecular coupling events that compensate enthalpic and entropic contribution of drug lead to similar free energies (∆G°) with different driving forces (∆H° or ∆S°). Our major observation from ITC experiments indicate that in presence of ILs or NaCl, binding affinities are reduced to almost half but overall interaction remains favorable. ∆G for DNA-EB interaction is around -7.8 kcal mol-1 which shows minor change in presence of ILs and NaCl which is around -7.2 kcal mol-1. ∆H changes for all the ILs are almost similar to buffer alone condition with minor decrease in case of Imidazolium based ILs (Table 1). This is because multimodal IL binding causes dehydration of the DNA through loss of water from minor groove, major groove and the spine of dehydration. The minor decrease in ∆G of ctDNA-EB interaction in ILs solution was due to unfavorable entropy which arises from the formation of more structured water cages in an attempt to rehydrate the DNA relative to that in the buffer condition whereas unfavorable ∆G of ctDNA-EB interaction in NaCl solution was due to less



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favorable enthalpy which arises from the interaction of Na+ to the phosphate backbone and not disturbing the overall hydrophobichydrophilic balance relative to that in the buffer condition. These observations indicate that effect of ILs on biomolecular interactions are majorly governed by different types of cationic groups in ILs.

CONCLUSION In this paper we have studied the effect of IL based solvents on small molecule- DNA interaction using ctDNA-EB as a model system. The effect of ILs on nucleic acid structure and stability was probed, wherein CD spectra suggest minor or no change in native B- form conformation for both group of ILs which is comparable with commonly used NaCl based solvents and in sync with previous observation for ILs. Moreover, ILs contribute to increased ctDNA stability, Bio-ILs i.e. choline based ILs show more stabilization in comparison to traditional Imidazolium based ILs. Combining observation from CD and UV, minor change in CD indicates that there is no effect of ILs on base stacking but in the presence of ILs, increased Tm indicates that ILs stabilizing effect on DNA helix is mediated through reducing repulsive coulombic interactions between the phosphates. EB exclusion result indicates that Imidazolium ILs are more effective in excluding EB than choline based ILs and NaCl. This difference in UV melting and EB exclusion experiments indicates that ILs binding of DNA varies depending on DNA structure and hydration. Comparison of thermodynamic parameters for ILs and NaCl lead to some interesting observations. ctDNA-EB interaction remains energetically favorable in presence of both NaCl and ILs, but contributing factors for favorability are antithetical in both. This behavior indicates about enthalpic-entropic compensation. In presence of IL, ctDNA-EB binding is governed through enthalpic contribution while in presence of NaCl, the governing factor is entropy with a lesser enthalpic contribution. This increased understanding of ILs’ thermodynamic contribution on ctDNA-EB interaction opens up new avenues for the use of ILs as alternative solvents for different DNA based biological applications. Thus, these reports suggest that IL based solvents are very similar to commonly used ionic solutions in molecular biological studies with unique properties like near zero vapor pressure, which prevents evaporation during reaction with small volumes. These findings suggest that IL based solvents may become the solvent of choice in future for DNA based micro fluidic devices or for the long term stability of DNA based digital storage technologies.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website Figure S1 showing Effect of IL on fluorescence intensity of EB without DNA, Normalized Fluorescence spectra at 600 nm for only EB in presence of different concentrations of ILs.

Acknowledgment This work was supported by Project BSC0124 (Project: CSIR-NCL-IGIB Joint Research Initiative: Interfacing Chemistry and Biology) from the Council of Scientific and Industrial Research (CSIR), Government of India.

NOTES The authors declare no competing financial interest.

REFERENCES ACS Paragon Plus Environment

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FIGURE LEGENDS Figure 1. CD spectra of ctDNA in presence of different concentrations ILs/NaCl ranging from 0 mM to 500 mM (100 mM intervals) were plotted to study the effect of IL/NaCl on ctDNA structure in presence of (a) NaCl (b) BMIM-Cl (c) BMIM –Br (d) choline lactate (e) choline acetate. CD signature of ctDNA at 275 nm in presence of NaCl (green circle) BMIM-Cl(blue triangle), BMIM-Br (cyan triangle), choline lactate (magenta square), Choline Acetate (yellow triangle) plotted in (f) to understand change in B-form DNA signature. Figure 2. UV Melting profile of ctDNA in presence of 100 mM (a) 200 mM(b) and 300 mM(c) ILs and NaCl. Only ctDNA (red square) ctDNA in presence of NaCl (green circle) BMIM-Cl (blue triangle), BMIM-Br (cyan triangle), choline lactate (magenta square), choline acetate (yellow triangle). Tm for ct DNA in presence of ILs/NaCl was plotted against IL/NaCl concentration to compare DNA stabilization effect of IL and NaCl (d). Figure 3. Fluorescence spectra of EB-DNA complex in presence of increasing concentration of (a) NaCl, (b) BMIM-Cl, (c) BMIMBr, (d) choline lactate and (e) choline acetate. Fluorescence of emission spectra of only EB and EB-DNA complex without ILs/NaCl are last and first spectra in each Figure, remaining spectra from 2nd spectra (100 mM) to 2nd last spectra (500 mM) represent increasing concentration of ILs/NaCl (from 100 mM, 200 mM, 300 mM, 400 mM, 500 mM). With increasing concentration of ILs/NaCl marked decrease in Fluorescence can be observed (reduction in fluorescence). (f) Normalized Fluorescence spectra at 600nm was plotted against different ILs/NaCl concentrations NaCl (green circle) BMIM-Cl (blue triangle), BMIM-Br (cyan triangle), or choline lactate (magenta square), choline acetate (yellow triangle) to compare EB exclusion capacity of each ILs and NaCl . Figure 4. ITC isotherms of EB-DNA binding in presence of 300mM ILs, NaCl and comparison with binding isotherms without ILs or NaCl. In each panel, the left plot is the baseline corrected experimental data (from Figure panel a- f). For the right panel plots (Figure panel g-i), results were converted to molar heats and plotted against the EB to DNA molar ration. All the ITC were performed in 10mM Sodium phosphate buffer (pH 7) with addition of equal concentration of ILs in DNA solution and EB solution. Buffer alone(a,g) In presence of NaCl (b,h), BMIM-Cl (c,i), BMIM-Br (d,j) , choline lactate(e,k) and choline acetate (f,i) . Figure 5. ∆∆ plots to compare thermodynamic signature between Ionic liquids and NaCl. This plot indicates ∆H, ∆S and ∆G changes with respect to buffer alone EB-DNA interaction condition. Here ∆∆H (white), ∆T∆S (grey) and ∆∆G (black) are plotted.


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Scheme 1 : (a) 1Butyl-3methylimidazolium Chloride (b) 1Butyl3methylimidazolium Bromide are two Imidazolium ring based ILs(c) 2-Hydroxyethyl-trimethylammonium-(+)-lactate (choline lactate ) and (d) CholineAcetate are two choline based ILs. Chemical structures were made using ChemDraw Ultra 8.0. Table 1. ITC experiments were performed in 10mM sodium phosphate buffer (pH 7) with addition of different ILs and NaCl respectively. Values of Kb, ∆H and N derived from fits of ITC data as described in text with indicated S.D. from experimental data.. Values of ∆G were calculated using standard formula ∆G = -RT ln(K) and Values of T∆S and ∆S were calculated using formula ∆G = ∆H – T∆S with indicated uncertainties reflecting maximum possible errors in ∆H and ∆G as propagated through this equation. Differential thermodynamic parameters (∆∆G, ∆∆H and ∆T∆S) are calculated by subtracting thermodynamic parameters of DNA-EB interaction in buffer condition from the thermodynamic parameters that are obtained under ILs and NaCl buffer conditions



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Figure 1 CD spectra

θ, mdeg

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Figure 2 UV melting

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Figure 3 EB exclusion:

Figure 4 : ITC



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Figure 5


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3

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Salts/ILs

Scheme 1:



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TABLE 1: ITC-Derived Binding Parameters at 25 °C for the ctDNA-EB at pH 7 with different Ionic liquids and NaCl


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Buffer condition

N

Kb × 105

∆H

T∆S

∆G

∆∆H

∆T∆S

∆∆G

mol-1

kcal mol-1

kcal mol-1

kcal mol-1

kcal

kcal mol-1

kcal mol-

mol-1

1

Buffer alone

0.37±0.01

5.79 ± 0.08

-9.3±0.25

-1.4±0.3

-7.8±0.01

0

0

0

NaCl

0.44±0.04

2.14 ± 0.09

-6.3±0.04

0.9±0.1

-7.2±0.03

3 ± 0.25

2.4± 0.31

0.6± 0.03

BMIM-Cl

0.43±0.03

2.45 ± 0.36

-8.73±0.12

-1.4±0.1

-7.3±0.08

0.6 ± 0.27

0.02± 0.31 0.5± 0.08

BMIM-Br

0.41±0.02

2.11 ± 0.07

-8.09±0.05

-0.8±0.1

-7.2±0.02

1.2 ± 0.25

0.6± 0.31

0.6± 0.02

Choline lactate

0.35±0.01

1.99 ± 0.03

-9.6±0.17

-2.4±0.2

-7.2±0.01

-0.3 ± 0.3

-0.9± 0.36

0.6± 0.01

Choline acetate

0.46±0.06

1.92 ± 0.21

-8.9±0.16

-1.8±02

-7.2±0.06

0.4 ± 0.29

-0.4± 0.36

0.6± 0.06

.

“For Table of Contents Only”



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216x121mm (150 x 150 DPI)


Influence of Ionic Liquids on Thermodynamics of Small MoleculeâDNA

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