Denaturation was examined for the first time in a ternary mixed solution of water/hydrophilic/ hydrophobic organic solvent using λ-DNA and a plasmid as models. The absorbance of λ-DNA and the plasmid at 260 nm gradually increased for several days up to 1.68 and 1.38 times the initial values, respectively, in a water/acetonitrile/ethyl acetate (15:3:2, volume ratio) mixed solution, whereas there was little change in a water/acetonitrile (15:3, volume ratio) mixed solution. The plasmid treated with the ternary mixed solution was also examined with agarose gel electrophoresis. These experimental data indicated that λ-DNA changed from a double helix structure to a single helix structure and that the plasmid partially transformed to generate a denaturation bubble in the structure. The new idea of using the ternary mixed solution first enabled the interaction of the hydrophobic organic solvent (e.g., ethyl acetate) molecule with the double helical structure of DNA, leading to specific slow-proceeding denaturation.
DNA denaturation and renaturation are important phenomena in a wide research area including biochemistry, analytical chemistry, and analytical science. DNA denaturation occurs when the hydrogen bonding between nucleotides is disrupted, and results in the separation of the strands. For example, denaturation of DNA due to high temperatures causes the disruption of the base pairs and the separation of the double-stranded helix into two single strands. Other than denaturation by heat, DNA can undergo denaturation through various chemical agents such as formamide, dimethyl sulfoxide, propylene glycol, and urea [
In addition, an area of partially separated DNA is known as the denaturation bubble [
In our previous paper [
Water was purified with an Elix 3 UV (Millipore Co.). All reagents used were commercially available and of analytical grade. Acetonitrile, ethyl acetate, and λ-DNA (0.35 μg・μL−1, MW 31,500,000, 48,502 bp, containing 10 mM Tris-HCl (pH 7.9) and 1 mM EDTA) were purchased from Wako Pure Chemical Industries, Ltd. Plasmid (0.12 μg・μL−1, 5446 bp, containing 10 mM Tris-HCl (pH 7.9) and 1 mM EDTA) was replicated from pcDNA3 (Invitrogen Co.).
Absorption analysis was carried out by an ultraviolet-visible spectrophotometer (JASCO Co., V-630). λ-DNA and plasmid original solutions were diluted with water, binary mixed solutions, and ternary mixed solutions to give fixed concentrations. Absorption spectra of λ-DNA and plasmid in the solutions were measured after every standing times. Absorbance at 260 nm in the spectra was examined in detail for DNA denaturation.
Agarose gel electrophoresis was performed using an electrophoresis system (Mupid-2Plus, Mupid. Com.) as follows. 15 μL of the water/acetonitrile/ethyl acetate mixed solution (15:3:2, volume ratio) containing plasmid (0.12 μg・μL−1), 5% glycerol, 0.09% SDS and 0.005% bromophenol blue was subjected to agarose gel electrophoresis. Plasmid was migrated in the gel containing 2.28 nM ethidium bromide with 100 V for ca. 40 min. After the migration, the gel was irradiated with ultraviolet rays to observe the separated plasmid bands.
We also examined the absorbance of λ-DNA with the ternary mixed solution as well as with pure water and a binary water/acetonitrile (15:3, volume ratio) solution for 120 h standing time (
values of 5 - 7 measurements. As shown in the figure, there was little change in the absorbance in both cases. The λ-DNA solution of the ternary mixed solution that was left for 48 h was diluted 5 times with water to obtain a component ratio of water/acetonitrile/ethyl acetate of 95:3:2, and then left for an additional 48 h. The molar absorption coefficient did not change, maintaining a constant value of ca. 0.80 L・cm−1・nmol−1 during the dilution. The denaturation of λ-DNA must be due to the presence of hydrophobic organic solvent, ethyl acetate, but the renaturation was not observed through the absorbance change under the present dilution treatment.
The increase in absorbance at 260 nm of λ-DNA in the water/acetoni- trile/ethyl acetate (15:3:2, volume ratio) mixed solution was compared to those in other ternary mixed solutions with the same component ratios. We determined their relative absorbances by defining the absorbance at 260 nm of λ- DNA (0.8 nM) in water/acetonitrile (15:3, volume ratio) for 48 h standing to be 1.0. The values of relative absorbance for water/acetonitrile/ethyl acetate, water/acetonitrile/chloroform, and water/acetonitrile/hexane were 1.60, 1.07, and 1.15, respectively. Those of water/acetonitrile/ethyl acetate, water/methanol/ ethyl acetate, and water/2-propanal/ethyl acetate were 1.60, 1.20, and 1.35, respectively. There was no absorbance change for water/methanol and water/2- propanal (15:3, volume ratio) mixed solutions. Thus, the hydrophobic organic solvent plays an important role in the denaturation that occurs with the ternary mixed solutions. The water/acetonitrile/ethyl acetate mixed solvent solution showed the largest change in absorbance at 260 nm of λ-DNA. Chemical and physical property of the ternary mixed solutions may cause other unique and interesting phenomena in DNA research area in future.
We examined the structure of the plasmid with agarose gel electrophoresis, but could not do that with λ-DNA because of its large molecular weight.
There was no evidence for the existence of a single-helix plasmid in the experiment; if a single-helix plasmid had existed, it would have migrated faster than the super-coiled one. However, as shown in
Denaturation was examined for the first time in the ternary water/hydrophilic/ hydrophobic organic solvent mixed solution using λ-DNA and a plasmid as models. The structure of λ-DNA changed from a double helix to a single helix and the plasmid formed a denaturation bubble in the water/acetonitrile/ethyl acetate (15:3:2, volume ratio) mixed solution. The ternary mixed solution first enabled contact of a hydrophobic organic solvent (e.g., ethyl acetate) molecule to the double helix of DNA to cause denaturation in the solution. DNA in the ternary mixed solution showed specific slow-proceeding denaturation, although it was difficult to compare and discuss other reported data, because of no data concerning DNA denaturation in ternary mixed solutions. DNA denaturation is well known and widely applied in DNA-related research areas. However, DNA denaturation also includes unclear aspects even now. DNA denaturation with interaction of hydrophobic organic solvent molecules in the ternary mixed solution is interesting as a new type of procedure and may provide clues to further clarify this process.
This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT) (No. 17H03083).
Ito, Y., Tsukagoshi, K. and Kobayashi, A. (2017) Denaturation of DNA in Ternary Mixed Solution of Water/Hydrophilic/Hydrophobic Organic Solvent. Journal of Analytical Sciences, Methods and Instrumentation, 7, 40-46. https://doi.org/10.4236/jasmi.2017.72004