Critical Assessment of the Interaction between DNA and Choline Amino Acid Ionic Liquids: Evidences of Multimodal Binding and Stability Enhancement

Abstract

Long-term storage and stability of DNA is of paramount importance in biomedical applications. Ever since the emergence of ionic liquids (ILs) as alternate green solvents to aqueous and organic solvents, their exploration for the extraction and application of DNA need conscientious understanding of the binding characteristics and molecular interactions between IL and DNA. Choline amino acid ILs (CAAILs) in this regard seem to be promising due to their non-cytotoxic, completely biobased and environment-friendly nature. To unravel the key factors for the strength and binding mechanism of CAAILs with DNA, various spectroscopic techniques, molecular docking, and molecular dynamics simulations were employed in this work. UV-Vis spectra indicate multimodal binding of CAAILs with DNA, whereas dye displacement studies through fluorescence emission confirm the intrusion of IL molecules into the minor groove of DNA. Circular dichorism spectra show that DNA retains its native B-conformation in CAAILs. Both isothermal titration calorimetry and molecular docking studies provide an estimate of the binding affinity of DNA with CAAILs ≈ 4 kcal/mol. The heterogeneity in binding modes of CAAIL-DNA system with evolution of time was established by molecular dynamics simulations. Choline cation while approaching DNA first binds at surface through electrostatic interactions, whereas a stronger binding at minor groove occurs via van der Waals and hydrophobic interactions irrespective of anions considered in this study. We hope this result can encourage and guide the researchers in designing new bio-ILs for biomolecular studies in future.

Figure 1

(a) Absorption spectra of free EB (10 μM) and EB-[Ch] [Gly] system varying the amount of ILs (50 mM and 100 mM) to a fixed concentration of EB (10 μM). (b) Absorption spectra of free DNA(60 μM) and DNA-[Ch] [Gly] system varying the amount of ILs (20 and 50 mM) to a fixed concentration of DNA (60 μM). (c) Absorption spectra of DNA-bound EB (10 μM EB, 60 μM DNA) in absence and presence of varying amounts of IL. (d) Melting curves of ct-DNA in buffer alone and 25, 50, and 100 mM [Ch] [Gly] IL. (e) CD spectra of 6.0 × 10^–5 mol/L DNA in phosphate buffer (10 mM, pH 7.0) and with increasing concentrations of [Ch][Gly] from 5 to 100 mM. (f) Emission spectra of free EB (10 μM), DNA-bound EB (10 μM, 60 μM DNA), and EB-DNA-[Ch] [Gly] system varying the amount of ILs (50 to 250 mM) to a fixed concentration of DNA-bound EB (10 μM, 60 μM DNA). (g) Stern–Volmer plot for [Ch] [Gly] IL. (h) Stern–Volmer plot for all three CAAILs. (i) Fluorescence decay profiles of free EB, EB-DNA in buffer, and EB-DNA system in the presence of 100, 200, and 300 mM [Ch] [Gly] IL.

Figure 2

ITC isotherms of EB-DNA binding in buffer and in the presence of 100 mM [Ch] [Gly] IL. (bottom) Experimental data. (top) Obtained by converting the results into molar heats and plotted against the EB to ct-DNA molar ratio.

Figure 3

Microscopic FE-SEM images (a) only 60 μM ct-DNA in 10 mM phosphate buffer, (b) 60 μM ct-DNA in the presence of 60 mM [Ch] [Gly], (c) 60 μM ct-DNA in the presence of 60 mM [Ch] [Ala], (d) 60 μM ct-DNA in the presence of 60 mM [Ch] [Pro].

Figure 4

Molecular docking (a) cholinium ion interaction at minor groove of DNA, (b) EB interaction at minor groove of DNA, (c) both EB and cholinium interact at minor groove of DNA.

Figure 5

Time evolution of (a) the minimum (proximal) distance between DNA and the CAAIL cation, where r_min < 0.3 nm signifies ligand being at the DNA surface, (b) number of water molecules within 0.3 nm of the CAAIL cation, where large values indicate fully solvated ligand in the bulk water phase, and lower values (<10) signify a desolvated buried state, and (c) number of hydrogen bonds between the DNA and CAAIL cation. (d) Time evolution of the nonbonded interaction energies between CAAIL cation and its surrounding. The Lennard-Jones (LJ) and electrostatic (Elec) interactions were calculated with everything else (including DNA, water, and ions) within a 2 nm cutoff radius of the cation. The black, red, and blue lines depict the LJ, electrostatic, and total energy, respectively. The data were locally averaged over 20 ps stretch for obtaining smoother curves.

Figure 6

Representative snapshots of dominant binding modes of CAAIL cation with DNA. (a) A state where the cation binds to the DNA surface through H-bonding interaction with the phosphate group. (b–d) Depictions of various possible H-bonded interactions while the cation is deeply buried in the minor groove.