Abstract
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.
Highlights
DNA denaturation and renaturation are important phenomena in a wide research area including biochemistry, analytical chemistry, and analytical science
In our previous paper [10], we reported that the peak shapes in the chromatograms of λ-DNA changed with a ternary water/hydrophilic/hydrophobic organic solvent mixed solution through the absorption detection at 260 nm, but the reason remained unclear
The change in absorbance of λ-DNA is due to change in structure from a double helix to a single helix, i.e., denaturation [11] [12] [13]
Summary
DNA denaturation and renaturation are important phenomena in a wide research area including biochemistry, analytical chemistry, and analytical science. Other than denaturation by heat, DNA can undergo denaturation through various chemical agents such as formamide, dimethyl sulfoxide, propylene glycol, and urea [1] These chemical denaturing agents lower the melting temperature by competing for hydrogen bond donors and acceptors with the pre-existing nitrogenous base pairs. An area of partially separated DNA is known as the denaturation bubble [6] [7] [8] [9]; non-covalent interactions between antiparallel strands in DNA can be partially broken in order to open the double helix when biologically important mechanisms such as DNA replication, transcription, DNA repair or protein binding are set to occur. DNA denaturation and the denaturation bubble were examined in a ternary water/hydrophilic/hydrophobic organic solvent solution using absorption spectroscopy, which was a challenge to know, for the first time, interaction between DNA molecules and hydrophobic organic solvent molecules. The hydrophobic organic solvent molecules in the ternary mixed solution could interact with DNA molecules in the solution to cause eccentric denaturation
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