Studies of the interaction mechanism between single strand and double-strand DNA with hydroxyapatite by microcalorimetry and isotherm measurements

Hydroxyapatite (HA) chromatography has been used extensively for the separation of single-strand deoxyribonucleic acids (ssDNA) and double-strand DNA (dsDNA). However, the details of the mechanism of ssDNA and dsDNA separation by hydroxyapatite are still not clear. In this study, we examined the effects of binding parameters (i.e. salt concentration, temperature and pH) and the effects of ssDNA and dsDNA base composition and sequence (i.e. GC content, length, secondary structure effects and GC or CG stacking rich, …) on the binding behavior. The affinities and binding amounts were obtained from equilibrium batch isotherm analysis, and the binding enthalpies were derived from isothermal titration calorimetry (ITC).Isotherm analysis reveals that electrostatic forces are the main driving force of dsDNA binding to HA. Due to hydrophobic bases and the negative charge of the phosphate backbone, the ssDNA molecule binds to HA with both hydrophobic and electrostatic interactions. Through thermodynamic analysis, we find that the adsorption enthalpies of both ssDNA and dsDNA are endothermic under all of the conditions studied and the dehydration step in the binding process plays a key role. The binding behavior responses to the binding parameters are discussed accordingly.