This study provides quantitative information about the kinetics of formation of a complex between DNA oligomers having 12 bp. The DNA dodecamers were designed in such a way as to avoid the formation of hairpins or slipped duplex structures within single strands. The hybridization was carried out employing stopped-flow techniques. The reaction was studied in different buffers (phosphate or cacodylate), in the presence and absence of Mg2+ ions, and at different temperatures. Under all conditions, the reaction followed second-order kinetics. The association rate constants were on the order of 106 M(-1) s(-1) and were found to increase with an increase in temperature. Both the rate constants and the positive activation energies of the two dodecamers, which differ only by the presence of the TAGG tetrad either at the 3'end or at the 5' end, turned out to be significantly different. The presence of Mg2+ ions had a profound influence on the kinetics of association of either compound by substantially decreasing the activation energy of the process. The dependence on sequence of the kinetics of hybridization was manifest in all parameters under all the experimental conditions.
Recently, we reported the kinetics of hybridization of cDNA dodecamers (Carrillo-Nava, E., Mejía-Radillo, Y., and Hinz, H.-J. Biochemistry 2008, 47, 13153-13157). In this study, we provide the thermodynamic reaction parameters of those dodecamers as well as a comparison with parameters for 24-mers designed from two identical dodecamers in tandem arrangement. The thermodynamic properties were determined by isothermal titration calorimetry (ITC), differential scanning microcalorimetry (DSC), and UV melting studies. On the basis of the results from our kinetic studies, fitting algorithms of DSC and UV melting profiles employed the two-state assumption for the duplex to a single strand dissociation reaction. The formation of both 12-mer and 24-mer duplexes is strongly enthalpy driven at all temperatures. At identical temperatures, the hybridization enthalpy of the 24-mer is within error limits twice that of the 12-mer. Duplex formation is always associated with a significant negative heat capacity change, ΔC(p), which, on a mass basis, is comparable to that observed for protein folding. Only a small part of the favorable reaction enthalpy appears as a standard Gibbs free energy change due to large compensating negative entropy changes linked to duplex formation. On the basis of the results of the present studies, it appears to be absolutely essential for a proper analysis of thermodynamic parameters of oligonucleotide hybridization reactions to combine low temperature ITC measurements of binding enthalpies with DSC and UV melting studies to obtain an accurate assessment of standard Gibbs energy changes or, equivalently, hybridization constants over a broad temperature range. The experimental thermodynamic parameters were compared with theoretical estimates based on nearest-neighbor approximations employing temperature-independent enthalpies. Good agreement between experimental and predicted ΔG° values is observed at ambient temperatures (20-30 °C), as long as helix formation is associated with small molar heat capacity changes. If the experimental ΔC(p) values determined by ITC are taken into account, significant deviations occur.
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