Pressure-Dependent Stability of Imidazolium-Based Ionic Liquid/DNA Materials Investigated by High-Pressure Infrared Spectroscopy

1-Butyl-3-methylimidazolium hexafluorophosphate ([C4MIM][PF6])/DNA and 1-methyl-3-propylimidazolium hexafluorophosphate ([C3MIM][PF6])/DNA mixtures were prepared and characterized by high-pressure infrared spectroscopy. Under ambient pressure, the imidazolium C2–H and C4,5–H absorption bands of [C4MIM][PF6]/DNA mixture were red-shifted in comparison with those of pure [C4MIM][PF6]. This indicates that the C2–H and C4,5–H groups may have certain interactions with DNA that assist in the formation of the ionic liquid/DNA association. With the increase of pressure from ambient to 2.5 GPa, the C2–H and C4,5–H absorption bands of pure [C4MIM][PF6] displayed significant blue shifts. On the other hand, the imidazolium C–H absorption bands of [C4MIM][PF6]/DNA showed smaller frequency shift upon compression. This indicates that the associated [C4MIM][PF6]/DNA conformation may be stable under pressures up to 2.5 GPa. Under ambient pressure, the imidazolium C2–H and C4,5–H absorption bands of [C3MIM][PF6]/DNA mixture displayed negligible shifts in frequency compared with those of pure [C3MIM][PF6]. The pressure-dependent spectra of [C3MIM][PF6]/DNA mixture revealed spectral features similar to those of pure [C3MIM][PF6]. Our results indicate that the associated structures of [C4MIM][PF6]/DNA are more stable than those of [C3MIM][PF6]/DNA under high pressures.


Introduction
Deoxyribonucleotide acid (DNA) molecules, which are double-helical biopolymers comprised of attached nucleotides, are well known to serve as genetic information carriers [1][2][3][4][5]. Because of the Watson-Crick interaction, that is, hydrogen bonding between nucleobase pyrimidine and purine, and base-base interaction (π-π stacking), DNA molecules can exist in stable conformations at ambient temperatures in cell nuclei [2,3]. The expected applications of DNA are far more than what current research can offer, because evidence increasingly suggests that DNA molecules are programmable in well-defined structures for 3D designing and topology [4,5]. Shih et al. [6] have discovered that ss-DNA can be configured into double-helical struts linked at the branched junctions, and two kinds of noncovalent motifs (double-crossover and paranemic-crossover struts) facilitate the formation of triangulated objects, such as tetrahedra or octahedra. Several studies [5][6][7][8][9] have also clarified that the specific associations and hydrophobic interactions make nucleic acid a promising candidate for the production of designable building blocks via self-assembly. In addition, various investigations show that the behaviors of self-assembly and electrostatic trapping of DNA can be used in drug delivery [8] and nanoelectronics [9].
Ionic liquids (ILs) are recognized as superb green solvents because of their low vapor pressure and recyclability, as well as several other unique characteristics [10][11][12][13][14][15], such as high electric conductivity High pressure (up to~2 GPa) was generated using a diamond anvil cell (DAC) with a diamond culet size of 0.6 mm. The DAC contained two type-IIa diamonds, which are suitable for mid-infrared (mid-IR) measurements. IR spectra were measured using a Fourier-transform (FT) spectrophotometer (Spectrum RXI, Perkin-Elmer, Naperville, IL, USA) equipped with a lithium tantalite detector. To enhance the intensity of passed infrared beam, a five-beam condenser was combined with the FT spectrometer. To eliminate the influence of absorption of the diamond anvils, the absorption spectra of the DAC were measured first and subtracted from those of the samples. A 0.25 mm thick Inconel gasket with a 0.3 mm diameter hole was prepared to hold the sample. To reduce the absorbance of the samples, transparent CaF 2 crystals were placed into the holes and compressed prior to inserting the samples. A resolution of 4 cm −1 (data point resolution of 2 cm −1 ) and 1000 scans were chosen for the high pressure data. Pressure calibration was performed following Wong's method [32,33]. The spectra of samples at ambient pressure were obtained by putting samples in a cell with two CaF 2 windows. To obtain the amount of water in the DNA mixtures (ca. 9 wt%), a moisture analyzer (MS-70, A&D Company, Tokyo, Japan) was used. Figure 1 shows the IR spectra of (a) pure [C 4 Figure 1a shows two imidazolium peaks at 3124 and 3169 cm −1 , which correspond to the vibrational absorption bands of C 2 -H and C 4,5 -H on the imidazolium ring, respectively [26][27][28]. The other three bands in the region of 2850-3000 cm −1 in Figure 1a are assigned to the alkyl C-H vibrational modes on the cation tail of pure [C 4 MIM][PF 6 ] [26][27][28]. The curve fitting and deconvolution of pure ionic liquids spectra were performed with Lorentzian peaks. In Figure 1b         The alkyl C-H bands in the range from 2850 to 3000 cm −1 are also blue-shifted because of the pressure increase ( Figure 3b). In agreement with our experimental results, several researchers [16,19,20,24,25] suggested that [C 4 [26][27][28] revealed that high pressure can enhance the cluster structure interaction and lead to a band frequency shift. The hydrogen-bond network of an IL cluster structure may be disrupted, as some added molecules (such as DNA) may disturb the associations of ILs or cut large aggregations to small pieces. With the further increase of pressure from 0.4 to 2.5 GPa (Figure 3b (Figure 3b). The alkyl C-H bands in the range from 2850 to 3000 cm −1 are also blue-shifted because of the pressure increase (Figure 3b). In agreement with our experimental results, several researchers [16,19,20,24,25] [26][27][28] revealed that high pressure can enhance the cluster structure interaction and lead to a band frequency shift. The hydrogen-bond network of an IL cluster structure may be disrupted, as some added molecules (such as DNA) may disturb the associations of ILs or cut large aggregations to small pieces. With the further increase of pressure from 0.4 to 2.5 GPa (Figure 3b-g (Figure 4A,B) Figure 4C show blue shifts at the pressure increase from ambient to 0.7 GPa and slight frequency shifts in the pressure range from 0.7 to 2.5 GPa. While the alkyl C-H absorption bands for the [C 4 MIM][PF 6 ]/DNA mixture ( Figure 4C) underwent blue shifts under pressures below 0.7 GPa, the alkyl C-H bands showed no significant frequency shifts at pressures above 0.7 GPa. The vibrational-band shifts of the C 4,5 -H and C 2 -H absorption bands of mixtures in Figure 4A,B show different trends in comparison with those for the alkyl C-H bands in Figure 4C. This indicates that local associations between imidazolium C-H (C 4,5 -H and C 2 -H) and DNA are dominant in the mixture, and the interactions between alkyl C-H and DNA are not sufficiently strong to fully disturb the alkyl C-H-anion interactions under high pressures. The band-shift differences under high pressures may be attributed to the differences in the interaction magnitudes of electrostatic association, hydrophobic interaction, and van der Waals force [1,16]. The IR spectra of [C 4 MIM][PF 6 ]/DNA (ambient and cycled back to ambient) are shown in Figure S2 (see Supplementary Materials), and the spectra are reversible upon pressure cycling. Pressure-induced reversible unfolding of biomolecules has drawn the attention of researchers [34]. Pressure denaturation leads to a more controlled perturbation to the structures of biomolecules than chemical or temperature denaturation. High-pressure NMR (with high resolution) may provide the sensitive approach in studies of the pressure-induced denaturation problems [34].

Results and Discussion
To investigate the interactions between DNA and ILs with various alkyl-chain lengths, combining [C 3 MIM][PF 6 ] and DNA may provide more hints on the effect of the DNA-IL association. Figure 5 shows the IR spectra of (a) pure [C 3 Figure 4A,B show different trends in comparison with those for the alkyl C-H bands in Figure 4C. This indicates that local associations between imidazolium C-H (C 4,5 -H and C 2 -H) and DNA are dominant in the mixture, and the interactions between alkyl C-H and DNA are not sufficiently strong to fully disturb the alkyl C-H-anion interactions under high pressures. The band-shift differences under high pressures may be attributed to the differences in the interaction magnitudes of electrostatic association, hydrophobic interaction, and van der Waals force [1,16]. The IR spectra of [C4MIM][PF6]/DNA (ambient and cycled back to ambient) are shown in Figure S2 (see Supplementary Materials), and the spectra are reversible upon pressure cycling. Pressure-induced reversible unfolding of biomolecules has drawn the attention of researchers [34]. Pressure denaturation leads to a more controlled perturbation to the structures of biomolecules than chemical or temperature denaturation. High-pressure NMR (with high resolution) may provide the sensitive approach in studies of the pressure-induced denaturation problems [34].        Figure 4A,B. The differences may be attributed to the stronger association between [C3MIM] + and [PF6] − caused by more symmetric and easier packing of [C3MIM] + than that of [C4MIM] + . Namely, cations with short alkyl side chain may favor the local cation-anion structures at high pressures. Cations with a longer alkyl side chain may lead to larger binding forces with DNA. This observation is consistent with the arguments reported in the literature [22,35]. In other words, the difference in alkyl side chain lengths may cause various effects on the stabilization of IL/DNA associations at high pressures. Figure 8C Figure 4A,B. The differences may be attributed to the stronger association between [C 3 MIM] + and [PF 6 ] − caused by more symmetric and easier packing of [C 3 MIM] + than that of [C 4 MIM] + . Namely, cations with short alkyl side chain may favor the local cation-anion structures at high pressures. Cations with a longer alkyl side chain may lead to larger binding forces with DNA. This observation is consistent with the arguments reported in the literature [22,35]. In other words, the difference in alkyl side chain lengths may cause various effects on the stabilization of IL/DNA associations at high pressures. Figure 8C shows the pressure dependence of alkyl C-H band shifts for pure [C 3

Conclusions
In this study, high-pressure measurements were performed to investigate the stabilization of DNA-IL associations. Pressure-dependent studies revealed that [C4MIM][PF6]/DNA association is

Conclusions
In this study, high-pressure measurements were performed to investigate the stabilization of DNA-IL associations. Pressure-dependent studies revealed that [C 4