Anion-Driven Influence of Tetrabutylammonium-Based Ionic Liquids on DNA Stability and Interaction Mechanisms

Abstract

Ionic liquids (ILs) have been widely used as alternative solvents for the stabilization, storage, and extraction of DNA. However, studies on the interaction between ammonium-based ILs and DNA, particularly focusing on the effect of anions, remain limited. Tetrabutylammonium (TBA) cation-based ILs with propanoate, bromide, glutamate, and threoninate anions were used to analyze IL–DNA interactions through UV–vis titrations, steady-state and time-resolved fluorescence, and molecular docking. The conformational stability and thermal stability of DNA in IL solutions were analyzed through circular dichroism spectroscopy and UV thermal studies, respectively. Viscosity measurements of the IL solutions were carried out to support the data obtained from UV thermal studies. The TBA cation displays multiple modes of interaction at the groove through electrostatic, hydrophobic, and hydrogen bonding. Among the studied anions, the propanoate anion exhibits significant hydrophobic interactions in addition to hydrogen bonding, whereas glutamate and threoninate primarily engage in hydrogen bonding. The difference in the effect of the ILs on DNA underscores the significant influence of the anions on IL–DNA interactions.

1. Introduction

Ionic liquids (ILs) have emerged as exceptionally effective solvents and cosolvents for stabilizing and preserving biomolecules, including nucleic acids, proteins, and enzymes. ILs are preferred over conventional solvents due to their distinctive characteristics, such as high chemical and thermal stability, low vapor pressure, task specificity, and reduced toxicity. − Specifically, in the field of biomolecules, IL media provide improved solubility, high stability, and extended lifespan, in addition to facilitating the recovery of both ILs and biomolecules. , Extensive studies show that the solubility and thermal stability of proteins can be improved in IL solvents as ILs interrupt aggregation and help maintain the activity of the protein. The application of ILs in biocatalysis is also promising as they act as stabilizing media for enzymes. The long-term stability of nucleic acids in ILs is influenced by interactions between ILs and nucleic acids. One of the key restrictions in using any system for biomolecules is toxicity. In the case of ILs, cytotoxic investigations suggest that a careful selection of anions and cations can mitigate this issue and serve as an effective approach. Low hydrophobicity, biocompatible ions, and nonfluorinated ions are some of the viable solutions to address the toxicity of ILs.

Nucleic acids have become formidable tools in the field of nanotechnology as their properties, such as sequence selectivity, find applications in developing sequence-sensing methods. Moreover, the implementation of DNA in nanotechnology extends to molecular transport devices, molecular motors, and molecular computing devices, in addition to potential applications in material science, life science, and more. DNA-associated research in aqueous media encounters a limitation with respect to the relative instability of DNA in water over extended periods at ambient temperature. Moreover, DNA extraction, the first step in any molecular diagnostic kit, is integral to the utilization of DNA in diverse scientific disciplines and can be a bottleneck as achieving high purity and separation from the cell matrix is a real challenge. Thus, the utilization of nonaqueous solvents, such as IL, as an extractantwhich has proven to stabilize and store DNAcan yield remarkable outcomes in DNA-based investigations.

The first evidence of the peculiarity of the existing interaction between IL and DNA was reported by Vijayaraghavan et al., where the exceptional solubility and stability of DNA for up to 1 year in choline-based ILs were demonstrated. The structural retention is correlated with the possibility of hydrogen bonding between the choline cation and the exterior of the DNA helix. Solvation and structural retention of DNA in neat ILs containing imidazolium, oxazolium, pyrrolidinium, pyrimidinium, and quaternary ammonium cations are reported. As an IL is composed of a cation and an anion, the general binding mechanism is such that the cation establishes electrostatic interaction with the phosphate group and is positioned near the DNA chain, while the anion forms hydrogen bonds with the bases. Choline-based ILs are well studied and exhibit a greater stabilizing effect on DNA compared to imidazolium-based ILs. This is attributed to the multiple hydrogen bonds formed by choline cations with ribose sugar and nucleobases, besides the phosphate backbone, in addition to groove binding. Multimodal binding of the choline cation with DNA was confirmed through molecular dynamic studies. Consequently, choline-based ILs demonstrated the capability to dissolve 8 wt % of DNA without any signs of degradation for up to 1 year, owing to the unique characteristics of the IL. Furthermore, successful regeneration of DNA was also achieved. Moreover, ILs aid in preventing hydrolytic damage by dehydrating the DNA and increasing its rigidity, thereby directly disrupting the DNA-DNase I interaction.

Although the B-DNA conformation was retained in the IL medium, the binding of imidazolium-based ILs induced a structural transformation of the DNA-IL complex from a coil to a globule form, where hydrophobic interactions between the alkyl chains of the IL and DNA bases played a major role. The effect of imidazolium ILs on the thermal stability of DNA was analyzed through multiwavelength UV resonance Raman technique that detected the selective binding of the IL cations to the guanine tracts of DNA and confirmed the dominance of groove binding over intercalation. Molecular docking studies of a few cations with DNA provided a comparison of the binding energies, in which imidazolium exhibited higher binding energies than morpholinium and cholinium ions. However, weaker binding energy is favored from the DNA extraction perspective as the back extraction will be feasible. The biocompatible morpholinium cation is found to predominantly bind in grooves and retain the structural integrity of DNA. Besides the type of cation and the interactions, pH, hydrophobicity, and concentration of the IL contribute to the structural changes in the DNA.

Ammonium-based ILs generally show high polarity and solvation capacity and have been reported by Singh et al. to solubilize 25% w/w of DNA, owing to the multiple binding possibilities offered by the cation and anion of the ILs. The mechanism involves hydrophobic and polar interactions with DNA, resulting in torsions and consequent changes in the 3D structure of DNA, leading to high solubility. One potential application of these interaction studies pertains to the utilization of ILs for DNA extraction. The highly efficient partitioning of DNA observed in ammonium IL-based aqueous biphasic system , represents a notable study that could be extended to real-system DNA extraction. The ILs that demonstrated effective DNA partitioning were composed of quaternary ammonium cations and biocompatible anions, such as carboxylates and amino acid-based anions. , Here, our objective is to exploit the interaction of DNA with tetrabutylammonium-based ILs containing biocompatible anions through various spectroscopic techniques, such as UV–vis absorption, steady-state fluorescence, time-resolved fluorescence, DNA melting study, and circular dichroism (CD), along with molecular docking.

2. Experimental Methods

2.1. Materials

Fish sperm DNA (dsDNA, DNA assay percentage greater than 99%, based on E_260 nm. The A_260/280 ratio is 1.86), ethidium bromide (purity: 95%), tetrabutylammonium hydroxide (40% aqueous solution), and tetrabutylammonium bromide (purity: ≥ 98%) were purchased from SRL Chemicals, India. l-Threonine (purity: 99%), l-glutamic acid (purity: 99%) were obtained from Spectrochem, and propanoic acid (purity: ≥ 99%) was sourced from Avra. The ultrapure water used for the preparation of the solutions for experiments was distilled using a Milli-Q3 purification system. The purity of the chemicals is as mentioned by the suppliers, and all chemicals were used as received.

DNA stock solution was prepared by adding the required amount of powdered fish sperm DNA in Millipore water and storing it at 4 °C for 1 day with gentle shaking to ensure complete dissolution. The concentration of DNA aqueous solutions was calculated using 6600 M^–1cm^–1 as the extinction coefficient value at 260 nm.

2.2. Instrumentation and Methods

Absorption spectra and UV melting studies were conducted on a PerkinElmer Lambda 365+ spectrometer with a Peltier temperature controller using a 1 cm path length quartz cuvette. UV melting spectra of DNA in Tris buffer and IL solutions were obtained at a wavelength of 260 nm in the temperature range of 30–100 °C at 2 °C intervals. Absorbance versus temperature graphs were plotted to determine the melting point, which is the midpoint of the transition curve. To identify the midpoint precisely, the first derivative of absorbance versus temperature graphs was plotted, and the peak of the curves represents the melting point of DNA. The viscosity of the IL solutions was measured using an Anton Paar microviscometer (Lovis 2000 ME) based on the rolling ball method.

The conformational analysis of DNA in water and in IL solutions was carried out using a Jasco J-815 CD spectrometer, and a quartz cuvette with a 1 cm path length was used for acquiring the spectra. The steady-state fluorescence was recorded with a Horiba Jobin-Yvon FluoroMax-4 spectrofluorometer, using a fixed 5 nm slit width for both excitation and emission. Time-resolved fluorescence was collected through a Horiba Jobin-Yvon time-correlated single-photon counting (TCSPC) spectrometer. A 450 nm nano-LED light source (fwhm = 3 ns) was used for the measurements. The instrument response function was recorded using Ludox AS40 colloidal silica. Decay data were analyzed using IBH software, and an acceptable χ² value was kept below 1.32 for a good fit. The average fluorescence lifetime (τ_avg) values were obtained using the following eq :

$${\tau }_{avg}=\frac{\sum _1^n{\beta }_i{\tau }_i^2}{\sum _1^n{\beta }_i{\tau }_i}$$ 1

where τ _i is the fluorescence lifetime and β _i is the corresponding normalized pre-exponential factor of component i.

2.2.1. Molecular Docking

The crystal structure of DNA (PDB: 1BNA, DD, 5′-d­(CGCGAATTCGCG)­2–3′) was downloaded from the Protein Data Bank , and the heteroatoms were removed using Discovery Studio. The ligand structurestetrabutylammonium cation, propanoate anion, glutamate anion, and threoninate anionwere optimized using Gaussian 16 (DFT, B3LYP, and 631G++(d,p) basis set). DNA and ligand files were prepared, and docking was performed using AutoDock 4.2 with the Lamarckian Genetic Algorithm. The grid size was set to 62, 74, and 116 in x-, y-, and z-directions, respectively, and the other parameters were set to default values. The docked conformation with the lowest energy was selected among the different poses. The mode of interactions between DNA and the ions was analyzed using Discovery Studio.

2.3. Synthesis and Characterization of Ionic Liquids

The synthesis of ILs, [TBA]­[Prop], [TBA]­[Glu], and [TBA]­[Thr] was carried out via a neutralization reaction between an acid or amino acid and tetrabutylammonium hydroxide. In the case of [TBA]­[Glu] and [TBA]­[Thr], aqueous [TBA]­OH was added to a slight excess of an equimolar amount of amino acid in aqueous media at room temperature and stirred continuously for 24 h. The solvent was removed using a rotary evaporator, and the excess amino acid was precipitated by adding a mixture of acetonitrile and methanol (9:1). The precipitate was filtered out, and the product was dried under vacuum. For the synthesis of [TBA]­[Prop], an equimolar amount of propanoic acid was added dropwise to aqueous [TBA]­OH under ice-cold conditions and stirred for 24 h at room temperature. The solvent was removed, and the product was dried under vacuum. The synthesized ILs were characterized by NMR and mass spectroscopy, as shown in Figures S1–S10, and the spectral data are given in the Supporting Information. NMR analysis was performed using a Bruker Avance Spectrometer (400 MHz) with D₂O solvent and internal standard TMS. A Quadrupole TOF LC/MS spectrometer was used for mass spectral analysis. The details of the ILs are listed in Table .

1. Structure, Name, and Abbreviation of Tetrabutylammonium-Based ILs.

3. Results and Discussions

3.1. UV–Vis Spectroscopy

Analysis and comparison of the absorption spectra of free DNA and IL-bound DNA apparently provide the simplest explanations for the DNA-IL interactions. The aqueous solution of DNA shows a broad UV band with an absorption maximum at 260 nm due to the chromophores in the purine and pyrimidine bases, and any change in the band in the IL-bound state emerges from possible interactions. The binding modes can be broadly classified into covalent and noncovalent, where covalent binding is identified by the observed hyperchromic effect with a redshift (bathochromic) in the absorption maxima. Other binding modes, namely electrostatic, intercalative, and groove binding, are noncovalent. Intercalation is the stacking interaction between a compound and DNA base pairs that results in hypochromism with a redshift. In the case of electrostatic interactions, hyperchromism is observed in the absorbance spectra corresponding to the conformational changes in DNA. The electrostatic interaction is expected between the cationic group of IL and phosphate groups in the DNA, and the interaction may result in DNA contraction. The absorbance changes observed in Figure a,c for [TBA]Br and [TBA]­[Prop] systems denote the possibility of electrostatic interaction, where the extent of the interaction is less. The minor hyperchromicity effects observed here, without any shifts, suggest weak interactions. However, increasing addition of [TBA]­[Glu] and [TBA]­[Thr] to DNA (Figure b,d) resulted in extended hyperchromicity with a moderate blueshift, denoting the presence of other types of interactions in addition to electrostatic binding. The observed high hyperchromicity also indicates partial uncoiling of the DNA helix, where the base pairs are exposed more, and consequently, the absorbance increases.

1.

Absorption spectra of DNA and DNA-IL systems with an increasing IL concentration to a fixed concentration of DNA (60 μM). (a) [TBA]­Br, (b) [TBA]­[Glu], (c) [TBA]­[Prop], and (d) [TBA]­[Thr].

The complexation of DNA with ethidium bromide (EB) dye in the presence of ILs is considered a reference for the analysis of DNA-IL interaction. EB is an intercalator; thus, hypochromicity with a redshift is observed in the absorption maxima of EB in the presence of DNA, as shown in Figure .

2.

Absorption spectra of free EB (10 μM), EB (10 μM)-DNA (60 μM), and DNA-bound EB with increasing [TBA]Br concentrations.

In the presence of IL, irrespective of the type, a blueshift (hypsochromic) is observed in the DNA-EB complex band due to the availability of free EB in the solution as a result of EB displacement by IL. The observed hypsochromism in the presence of [TBA]Br is applicable to other ILs as well, as seen in Figure S11. The changes observed in the system containing EB, DNA, and IL may not exactly represent interactions between IL and DNA since there is a possibility of EB-IL interactions. This is verified through control experiments where the absorption spectra of EB with all the ILs were measured, as shown in Figure S12. In the presence of ILs, the absorbance curve of EB exhibited hyperchromism. The absorbance peak was enhanced as the IL concentration increased in the studied systems, except in the case of [TBA]­Br. Notably, [TBA]­[Glu] has extensive ground-state interaction with EB.

3.2. Steady-State and Time-Resolved Fluorescence

Fluorescence spectroscopy aids in elucidating ligand-DNA interactions, but here, the ILs and DNA are not fluorescent; thus, the common fluorophore EB has to be incorporated. EB in water shows low fluorescence intensity as the water medium quenches the excited-state molecules by proton transfer to water molecules. The fluorescence intensity of DNA-bound EB is higher than that of free EB, corresponding to intercalation and reduced proton transfer. The introduction of a quencher molecule, IL, diminishes the DNA-EB fluorescence owing to the displacement of EB by IL, and the extent of fluorescence quenching reveals the extent of binding between IL and DNA. Figure shows the quenching of DNA-EB high-intensity fluorescence with increasing concentrations of IL. The electrostatic interaction between the cationic group of IL and the phosphate group of DNA results in DNA compaction, preventing EB intercalation.

3.

Fluorescence spectra of free EB (10 μM), DNA-EB complex (10 μM, 60 μM DNA), and EB-DNA with increasing concentration of ILs, (a) [TBA]­Br, (b) [TBA]­[Glu], (c) [TBA]­[Prop], and (d) [TBA]­[Thr].

To quantitatively assess the quenching, the measured intensity values were fitted to the Stern–Volmer eq (eq ) to calculate the fluorescence quenching constant, K _SV (Figure )­

$$F_0/F=1+K_{SV}[Q]$$ 2

4.

Stern–Volmer plot for the [TBA]-based ILs.

where K _q and K _SV are the quenching rate constant and the stern Volmer constant, respectively, τ₀ denotes the average lifetime of a fluorophore, and F ₀ and F represent the intensities without and with a quencher in the system. The obtained K _SV values are 2.402, 2.215, 2.137, and 8.574 M^–1 for [TBA]­Br, [TBA]­[Glu], [TBA]­[Prop], and [TBA]­[Thr] respectively, as the quenchers. The quenching constant values are different for the ILs, indicating the role of anions in the binding.

Here, as there is an IL-EB interaction present in the system, as evidenced by the absorbance studies, quenching constants may not correspond to quenching efficiency. To confirm IL-EB interactions in the excited state, control experiments were performed (Figure S13) and intensity enhancement of EB in the presence of ILs was observed as nonaqueous solvents reduce the rate of proton transfer in EB. Although this may affect the intensity changes in the EB exclusion studies, displacement of EB by IL is indeed valid, as is clear from UV studies (Figure ) and fluorescence analysis (Figure ).

The fluorescence decay profiles of EB-DNA in the presence of varying IL concentrations were measured and are shown in Figure for all the IL systems. The calculated decay parameters obtained from biexponential fits (eq ) for the analyzed systems are included in Table .

$$I(t)=a_1\exp (-t/{\tau }_1)+a_2\exp (-t/{\tau }_2)$$ 3

5.

Fluorescence decay profiles of EB, EB-DNA, and EB-DNA system with increasing concentration of ILs, (a) [TBA]­Br, (b) [TBA]­[Glu], (c) [TBA]­[Prop], and (d) [TBA]­[Thr].

2. Fluorescence Decay Parameters (Lifetimes in Nanoseconds) of Free EB, EB-DNA in Buffer, and EB-DNA System in the Presence of Increasing IL Concentrations.

Sample	τ1	α1	τ2	α2	χ2
EB (10 μM)	1.60 × 10–9	100			1.03
EB (10 μM)-DNA (60 μM)	1.81 × 10–9	46.23	1.25 × 10–8	53.77	1.17
EB-DNA-[TBA]Br
20 mM IL	1.80 × 10–9	58.72	1.40 × 10–8	41.28	1.02
50 mM IL	1.82 × 10–9	64.82	1.48 × 10–8	35.18	1.21
100 mM IL	1.90 × 10–9	70.61	1.56 × 10–8	29.39	1.03
150 mM IL	1.99 × 10–9	74.51	1.60 × 10–8	25.49	1.12
200 mM IL	2.08 × 10–9	76.89	1.63 × 10–8	23.11	1.17
250 mM IL	2.18 × 10–9	78.48	1.68 × 10–8	21.52	1.07
EB-DNA-[TBA][Glu]
20 mM IL	1.75 × 10–9	55.29	1.40 × 10–8	44.71	1.25
50 mM IL	1.77 × 10–9	58.94	1.46 × 10–8	41.06	1.17
100 mM IL	1.84 × 10–9	64.83	1.54 × 10–8	35.17	1.00
150 mM IL	1.87 × 10–9	66.18	1.58 × 10–8	33.82	1.01
200 mM IL	1.96 × 10–9	68.11	1.59 × 10–8	31.89	1.20
250 mM IL	2.02 × 10–9	70.16	1.59 × 10–8	29.84	1.25
EB-DNA-[TBA][Prop]
20 mM IL	1.84 × 10–9	53.35	1.37 × 10–8	46.65	1.17
50 mM IL	1.83 × 10–9	58.57	1.43 × 10–8	41.43	1.04
100 mM IL	1.87 × 10–9	65.42	1.49 × 10–8	34.58	1.23
150 mM IL	1.90 × 10–9	66.53	1.54 × 10–8	33.47	1.17
200 mM IL	1.99 × 10–9	69.22	1.59 × 10–8	30.78	1.16
250 mM IL	2.03 × 10–9	70.81	1.61 × 10–8	29.19	1.21
EB-DNA-[TBA][Thr]
20 mM IL	1.72 × 10–9	63.72	1.28 × 10–8	36.28	1.21
50 mM IL	1.69 × 10–9	70.59	1.37 × 10–8	29.41	1.25
100 mM IL	1.75 × 10–9	78.48	1.47 × 10–8	21.52	1.11
150 mM IL	1.78 × 10–9	83.73	1.48 × 10–8	16.27	1.27
200 mM IL	1.80 × 10–9	86.26	1.44 × 10–8	13.74	1.05
250 mM IL	1.83 × 10–9	88.32	1.43 × 10–8	11.68	1.19

where τ₁ and τ₂ are lifetime components and a ₁ and a ₂ are amplitudes associated at t = 0.

The free EB in aqueous solution shows single exponential decay with a 1.6 ns lifetime, and upon binding with DNA, biexponential decay is observed, corresponding to lifetimes of 18.5 and 1.25 ns. When ILs are added successively to the EB-DNA system, the amplitude of the initial component associated with EB increases, while the newly formed decay component diminishes due to the exclusion of EB from the EB-DNA complex by IL. This result is consistent with findings from fluorescence studies. The extent of EB exclusion is found to be maximum in the presence of a higher concentration of [TBA]­[Thr], as observed in the fluorescence analysis. However, the lifetime values of EB increase as the concentration of the quencher (IL) increases. This can be explained by control experiments, where the fluorescence decay of EB in the presence of IL was measured, as shown in Figure S14, with the lifetime values presented in Table S1. Table S1 clearly shows the enhancement in the lifetime values of EB with increasing IL concentration, which leads to the observed increase in the lifetime values of the EB-DNA-IL system, as shown in Table . [TBA]­[Glu] has extended the excited-state interaction with EB, as observed in UV studies (Figure S12). Consequently, a new lifetime component with a relatively low amplitude, corresponding to the EB-IL complex, exists in the presence of increased IL concentrations.

The results from absorption spectra, steady-state spectra, and time-resolved fluorescence studies are well aligned with each other and indicate extended interaction between [TBA]­[Thr] and DNA, which may cause partial degradation of DNA. Even though [TBA]­[Glu] has strong interactions with DNA, it is not reflected in the studies involving the EB intercalator as [TBA]­[Glu] has a comparatively higher affinity for EB among the studied ILs. However, [TBA]Br and [TBA]­[Prop] exhibit weak but significant interactions with DNA, and the preliminary studies indicate that these ILs do not induce any major changes in the DNA structure.

3.3. Circular Dichroism

The conformational integrity of DNA in the studied ILs can be analyzed through CD spectroscopy, where characteristic CD spectra of DNA constitute a positive band at 275 nm and a negative band at 245 nm, corresponding to base stacking and the helical nature of DNA, respectively. The base pairs are not inherently chiral, but the chirality in the attached sugar moieties is responsible for the band at 275 nm. The CD spectra of DNA in the presence of successive additions of lower concentrations of ILs, from 4 to 20 mM, and higher concentrations of ILs, from 20 to 250 mM, are shown in Figures and , respectively. The absence of any major deviation in the characteristic spectra of DNA under the influence of lower concentrations of all the studied ILs indicates the stability of the B form of DNA. The DNA in higher concentrations of [TBA]Br and [TBA]­[Prop] is also conformationally intact, where the minor deviations in the amplitude of the signal can be attributed to minor alterations in base packing and helicity as a result of the electrostatic and hydrophobic interactions between DNA and IL. The absence of intercalation is apparent from Figure , and it complies with the small perturbations in the CD spectra. Thus, the IL interaction might be through the grooves of DNA, corresponding to conformational stability. Whereas in the case of [TBA]­[Thr] IL, a loss of the helical nature of DNA is observed at higher concentrations of the IL (above 200 mM), which is in agreement with the observed extended interactions from the EB exclusion studies. [TBA]­[Glu] also induces conformational destabilization of DNA as the concentration of IL increases above 100 mM. A comparison of the studied ILs indicates that [TBA]Br and [TBA]­[Prop] help maintain the conformational stability of DNA, while [TBA]­[Glu] and [TBA]­[Thr] destabilize DNA at higher concentrations.

6.

CD spectra of DNA in water and in 4–20 mM aqueous IL solutions, (a) [TBA]­Br, (b) [TBA]­[Glu], (c) [TBA]­[Prop], and (d) [TBA]­[Thr].

7.

CD spectra of DNA in water and in 20–250 mM aqueous IL solutions (a) [TBA]Br (b) [TBA]­[Glu] (c) [TBA]­[Prop] and (d) [TBA]­[Thr].

3.4. Thermal Melting Study

The UV thermal experiment was conducted to investigate the thermal stability of DNA under different conditions, in both the absence and presence of ILs. Absorbance measurements of DNA at 260 nm with respect to an increase in temperature were performed, and the obtained results are shown in Figure . Melting of DNA causes the unstacking of the bases, which leads to denaturation, where double-stranded DNA changes into single-stranded DNA. Thus, with the elevation in temperature, the absorbance of DNA increases (hyperchromism), and the absorbance versus temperature plots are called melting curves. The melting temperature can be determined from the first derivative of the melting curves, and the graphs are presented in Figure S15. Figure shows the melting curves of DNA in Tris-HCl buffer in the absence and presence of varying concentrations of [TBA]­[Prop]. The temperature at which the DNA undergoes melting in the absence of ILs is determined to be 69.5 °C, whereas the melting temperature increases to 77 °C in the presence of 20 mM [TBA]­[Prop]. However, high concentrations of [TBA]­[Prop] do not increase the thermal stability of DNA as the melting points in the presence of 50 and 100 mM IL concentrations are found to be 61.4 and 44.4 °C, respectively. DNA stabilization at higher IL concentrations is influenced by ion-specific effects as the individual IL ions contribute significantly. , The melting curves of DNA in the presence of [TBA]­[Br], [TBA]­[Thr], and [TBA]­[Glu] are shown in the Figure S16, where it is clear that the ILs are not stabilizing the DNA structure at elevated temperatures. The presence of [TBA]­[Glu] in higher concentrations induces significant changes in DNA conformation at both ambient and elevated temperatures, as evidenced by CD and UV thermal studies.

8.

UV melting curves of DNA in Tris-HCl buffer and various concentrations of [TBA]­[Prop].

3.5. Viscosity Measurements

The thermal melting of DNA depends on the viscosity of the solvent as the melting point of DNA decreases when the viscosity of the solvent increases. The viscosity dependence is due to the reversible melting nature of DNA, and, during the melting process, DNA migrates in the medium to find stable base-pairing partners. Thus, to verify the trend observed in the thermal melting exerted by the IL solvents, the viscosity effect also has to be accounted for. The dynamic viscosity values of the solvents at 30 °C were measured and are listed in Table . The viscosity values show that [TBA]­[Prop] solutions are comparatively more viscous than the other IL solutions. However, enhanced thermal melting was observed in [TBA]­[Prop], indicating favorable interactions between [TBA]­[Prop] and DNA, resulting in the stabilization of DNA. Additionally, the IL [TBA]­[Prop] has a minimum inhibitory concentration value of 1970 mg/L and an EC₅₀ value of 1640 mg/L, which is considered “practically harmless” in terms of the toxicity of the IL, as previously reported.

3. Viscosity Values of IL Solutions Used for UV Thermal Studies.

IL	Concentration in buffer (mM)	Viscosity (mPaS)	Tm (°C)
[TBA]Br	20	1.097	62.7
	50	1.134	57.6
[TBA][Glu]	20	1.098	45.1
[TBA][Prop]	20	1.102	77.0
	50	1.140	61.0
	100	1.214	44.1
[TBA][Thr]	20	1.092	60.4
	50	1.12	53.9

3.6. Molecular Docking Studies

As the ILs selected here have a common cation and different anions, the interaction energy between DNA and the ions was calculated using docking to compare the effect of the anions. The docked structures (Figure a) revealed the minor groove binding of the tetrabutylammonium cation, which is associated with the absence of major deviations in the structure of DNA in IL media. The changes observed in the amino acid-based ILs are attributed to the anionic effect. The binding energies of the cation–anion interactions with DNA are included in Table . As expected, the binding energy of the TBA cation is higher than that of the anions due to the electrostatic interaction established between the positively charged ammonium ion and the negatively charged phosphate groups of DNA. Specifically, the cation binds to the A-T-rich regions of the DNA through electrostatic interactions, hydrogen bonding, and hydrophobic interactions. The hydrophobic interactions are primarily between the π-orbitals of the base pairs and the alkyl groups of the cation. The different types of interactions are represented in Figure b for the conformation with the highest binding energy. The electrostatic contribution is insignificant in this particular conformation. One of the docked positions showing all types of interactions is provided in Figure S17 for reference.

9.

Docking images representing (a) TBA cation in the DNA groove. (b) 2D diagram of binding showing the interactions for the most stable docking position.

4. Interaction Energies (in kcal·mol^–1) Obtained from the Docking Study of the Cations and Anions with 1BNA.

Cation/Anion	Binding energy	van der Waals + Hydrogen bonding + Desolvation energy	Electrostatic energy
[TBA]+	–4.43	–7.31	–0.70
[Glu]¯	–4.20	–5.46	–0.52
[Prop]¯	–3.26	–3.49	–0.07
[Thr]¯	–4.14	–5.19	–0.14

The free energy of binding is obtained as −4.43 kcal/mol, corresponding to a binding constant of 1.75 × 10³ L·mol^–1. Table shows the energy of different interactions, where desolvation energy represents the hydrophobic interaction energy between the alkyl chains of the ions and the hydrophobic moieties of DNA. Similar to previous observations in the case of imidazolium and morpholinium cations, the electrostatic contribution to the total binding energy is less compared to other types of interactions. ,

When comparing the anions, the glutamate anion, [Glu]¯, exhibits the highest binding order through conventional and nonconventional (C–H) hydrogen bonding with the bases. The lowest affinity of the propanoate anion, [Prop]¯, is promising in terms of the back extraction of the ILs from the IL-DNA complex. The difference in the interactions among the studied anions is in agreement with experimental studies, where [TBA]­[Prop] showed comparatively less interaction. All three anions formed hydrogen bonds with DNA, where [Prop]¯ displayed additional hydrophobic interaction between the π-orbitals of nitrogen bases and the alkyl chain of the anion. The diagrams representing anion-DNA interactions are provided in Figures S18–S20.

The integrated analysis highlights that [TBA]Br and [TBA]­[Prop] primarily interact with DNA through weak electrostatic forces, as indicated by minor hyperchromicity without spectral shifts, while DNA remains structurally intact across varying concentrations. In contrast, [TBA]­[Glu] and [TBA]­[Thr] exhibit stronger interactions, combining electrostatic and hydrogen bonding, leading to significant hyperchromicity and partial DNA uncoiling. Fluorescence quenching studies further confirm that [TBA]­[Thr] causes maximum displacement of EB, aligning with the observed DNA destabilization at higher concentrations. [TBA]­[Glu] also induces DNA conformational changes, although its affinity for EB complicates interpretation in fluorescence quenching assays. Comparing anions, [Glu]¯ shows the strongest binding via conventional and nonconventional hydrogen bonds, while [Prop]¯ displays the weakest, promising better reversibility. Together, the spectroscopic techniques consistently demonstrate a gradient of interaction strengths, ranging from weak reversible binding in [TBA]­[Prop] and [TBA]Br to stronger destabilizing effects in [TBA]­[Glu] and [TBA]­[Thr].

4. Conclusions

Interaction studies of DNA with tetrabutylammonium cation-based ILs containing bromide, glutamic acid, propanoic acid, and threonine anions were carried out through spectroscopic analysis. UV–vis studies revealed electrostatic, hydrophobic, and groove-binding interactions, whereas the EB exclusion assay through fluorescence showed that ILs can displace EB from DNA as a result of these interactions. The absorbance spectra of DNA in the presence of different IL concentrations suggest the comparatively weaker binding of [TBA]Br and [TBA]­[Prop] through electrostatic interactions, whereas [TBA]­[Thr] and [TBA]­[Glu] displayed stronger binding through multiple modes of interaction. The excited-state interaction study required the presence of a dye, ethidium bromide (EB), to understand the system, and the results are similar to those of the ground-state interaction, except [TBA]­[Glu] did not show extended interaction here. This is attributed to the strong EB–[TBA]­[Glu] binding, as suggested by the control studies, which were performed to evaluate the ground-state and excited-state interactions between EB and ILs. EB showed enhanced fluorescence in the presence of ILs. EB exclusion studies were supported by lifetime analysis, and the IL with the threonine anion showed a more prominent interaction than other studied anions. This finding underscores the influence of anions on DNA binding. Conformational analysis of DNA using CD spectroscopy revealed partial degradation of DNA at higher concentrations of [TBA]­[Thr] and [TBA]­[Glu], likely due to the strength of their interactions. In contrast, other ILs demonstrated the ability to provide structural stability to DNA. The higher binding affinity is not favorable in terms of the stability of DNA, as recommended by the CD spectrum results. The conformational stability of DNA was unaffected at lower IL concentrations for all the studied ILs; nevertheless, higher concentrations of [TBA]­[Glu] and [TBA]­[Thr] induced partial degradation of the double helix. Regarding the thermal stability of DNA, lower concentrations of [TBA]­[Prop] showed promising results, while all the other systems exhibited unfavorable outcomes. Viscosity measurements of IL solutions rule out solvent effects on thermal melting, with [TBA]­[Prop] enhancing DNA stability despite higher viscosity. Molecular docking studies were conducted to elucidate the interaction modes and points. The TBA cation displays multiple modes of interaction at the groove through electrostatic interactions, hydrophobic interactions, and hydrogen bonding. The propanoate anion shows significant hydrophobic interactions in addition to hydrogen bonding, whereas glutamate and threoninate primarily engage in hydrogen bonding. The strength of binding suggested by the docking studies is similar to that of experimental studies. Additionally, the comparatively lower binding energy of the propanoate anion explains the enhanced DNA stability in [TBA]­[Prop]. The difference in the influence of the ILs on DNA underscores the significant role of the anions in IL-DNA interactions. The study emphasizes the importance of assessing these interactions to elucidate IL-assisted DNA processes.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsphyschemau.5c00015.

• ¹H NMR, ¹³C NMR, and mass spectra of the synthesized ILs are presented in Figures S1–S10; absorption, fluorescence, and fluorescence decay spectra of EB are provided in Figures S11–S14. UV melting curves and their derivatives are shown in Figures S15 and S16; and diagrams depicting the docked conformations are included in Figures S17–S20 (DOCX)

CRediT: Kadavathu Kambrath Athira conceptualization, data curation, formal analysis, investigation, methodology, software, validation, writing - original draft; Ramesh L. Gardas conceptualization, funding acquisition, methodology, project administration, resources, supervision, writing - review & editing.

The authors declare no competing financial interest.