Insights on the DNA Stability in Aqueous Solutions of Ionic Liquids

Abstract

Deoxyribonucleic acid (DNA) carries the genetic information essential for the growth and functioning of living organisms, playing a significant role in life sciences research. However, the long-term storage and preservation of DNA, while ensuring its bioactivity, are still current challenges to overcome. In this work, aqueous solutions of ionic liquids (ILs) were investigated as potential preservation media for double stranded (dsDNA). A screening of several ILs, by combining the cholinium, tetrabutylammonium, tetrabutylphosphonium, and 1-ethyl-3-methylimidazolium, cations with the anions bromide, chloride, dihydrogen phosphate, acetate, and glycolate, was carried out in order to gather fundamental knowledge on the molecular features of ILs that improve the dsDNA stability. Different IL concentrations and the pH effect were also addressed. Circular dichroism (CD) spectroscopy was used to evaluate the conformational structure and stability of dsDNA. IL-DNA interactions were appraised by UV-Vis absorption spectrophotometry and ³¹P nuclear magnetic resonance (NMR) spectroscopy. The results obtained demonstrate that pH has a significant effect towards the dsDNA stability. Amongst the ILs investigated, cholinium-based ILs are the most promising class of ILs to preserve the dsDNA structure, in which electrostatic interactions between the cholinium cation and the DNA phosphate groups play a significant role as demonstrated by the ³¹P NMR data, being more relevant at higher IL concentrations. On the other hand, the denaturation of dsDNA mainly occurs with ILs composed of more hydrophobic cations and able to establish dispersive interactions with the nucleobases environment. Furthermore, the IL anion has a weaker impact when compared to the IL cation effect to interact with DNA molecules. The experimental data of this work provide relevant fundamental knowledge for the application of ILs in the preservation of nucleic acids, being of high relevance in the biotechnology field.

Keywords: DNA; interactions; ionic liquids; native conformation; nucleic acid; stability.

FIGURE 1

Chemical structures of the ILs used in this work: (A) [N₄₄₄₄]Br; (B) [P₄₄₄₄]Br; (C) [C₂C₁im]Br; (D) [N_111(2OH)]Br; (E) [N_111(2OH)]Cl; (F) [N_111(2OH)][Ac]; (G) [N_111(2OH)][DHP]; (H) [N_111(2OH)][Gly].

FIGURE 2

CD spectra regarding the ellipticity, θ, of 0.01 g dm⁻³ of β-DNA (from salmon testes) as a function of wavelength, λ, in 10 mM of Tris–HCl buffer (pH ≈ 7.2) aqueous solutions at different concentrations of bromide-based ILs: (A) 5 wt%; (B) 15 wt%; (C) 30 wt%. () only buffer; () [N_111(2OH)]Br; () [N₄₄₄₄]Br; () [C₂C₁im]Br; () [P₄₄₄₄]Br.

FIGURE 3

CD spectra regarding the ellipticity, θ, of 0.01 g dm⁻³ of β-DNA (from salmon testes) as a function of wavelength, λ, in 10 mM of Tris–HCl buffer (pH ≈ 7.2) aqueous solutions at different concentrations of cholinium-based ILs: (A) 5 wt%; (B) 15 wt%; (C) 30 wt%. () only buffer; () [N_111(2OH)]Br; () [N_111(2OH)]Cl; () [N_111(2OH)][Ac]; () non-buffered [N_111(2OH)][DHP]; () buffered [N_111(2OH)][DHP]; () [N_111(2OH)][Gly].

FIGURE 4

Schematic representation of (A) DNA dissolution process with the increase of buffer concentration (increase of pH value) and (B) CD spectra regarding the ellipticity, θ, of 0.01 g dm⁻³ of β-DNA (from salmon testes) as a function of wavelength, λ, in 1000 mM Tris–HCl buffer (pH ≈ 7.2). () only buffer; () 5 wt% of [P₄₄₄₄]Br; () 15 wt% of [P₄₄₄₄]Br; () 30 wt% of [P₄₄₄₄]Br.

FIGURE 5

Absorption spectra of β-DNA (from salmon testes) as a function of wavelength, λ, in 10 mM of Tris–HCl buffer (pH ≈ 7.2) aqueous solutions at different concentrations of IL. (A) 5 wt% of bromide-based ILs: () only buffer; () [N_111(2OH)]Br; () [C₂C₁im]Br; () [N₄₄₄₄]Br; () [P₄₄₄₄]Br; (B) 5 wt% of cholinium-based ILs. () only buffer; () [N_111(2OH)]Br; () [N_111(2OH)][Ac]; () non-buffered [N_111(2OH)][DHP]; () [N_111(2OH)]Cl; () [N_111(2OH)][Gly]; (C) 30 wt% of bromide-based ILs: () only buffer; () [N_111(2OH)]Br; () [C₂C₁im]Br; () [N₄₄₄₄]Br; (D) 30 wt% of cholinium-based ILs. () only buffer; () [N_111(2OH)]Br; () [N_111(2OH)][Ac]; () non-buffered [N_111(2OH)][DHP]; () [N_111(2OH)]Cl; () [N_111(2OH)][Gly].

FIGURE 6

³¹P NMR intensity peaks of DNA as a function of dsDNA ellipticity at 245 nm. (A) IL at 5 wt%; (B) IL at 30 wt%.

FIGURE 7

³¹P NMR intensity peak of DNA as a function of dsDNA ellipticity at 280 nm. (A) IL at 5 wt%; (B) bromide-based ILs at 30 wt%; (C) cholinium-based ILs at 30 wt%.