83 Application of differential scanning calorimetry to measure the differential binding of ions, water, and protons in the unfolding of DNA molecules

Abstract

The overall stability of DNA molecules globally depends on base-pair stacking, base pairing, polyelectrolyte effect, and hydration contributions. In order to improve our understanding of the role of ions, water, and protons in the stability and melting behavior of DNA structures, we report an experimental approach to determine the differential binding of ions (Δn ion), water (Δn W), and protons (Δn H+) in the helix-coil transition of DNA molecules. A combination of differential scanning calorimetry (DSC) and temperature-dependent UV and CD spectroscopic techniques to investigate the unfolding of a variety of DNA molecules: S.T. DNA, two dodecamers, one undecamer, nine short hairpins as a function of the GC content of their stem, and two triplexes. We determine complete thermodynamic profiles, including all the three linking numbers, for the unfolding of each molecule. The CD spectra indicated that all molecules adopted the B-conformation at low temperatures. Thermodynamic profiles obtained from the DSC curves indicate that the favorable folding of each molecule results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, and negligible heat capacity effects. UV and DSC melting curves as a function of salt, osmolyte, and proton concentrations yielded releases of ions, water, and protons (for the triplex with C+GC base triplets). Therefore, the favorable folding of each DNA molecule results from the formation of base-pair stacks and uptake of water and counterions. The thermodynamic data will be discussed in terms of the effects of DNA length, loop contributions and type of water molecules.