Temperature dependence and sequence specificity of DNA triplex formation: An analysis using isothermal titration calorimetry

We have investigated the thermodynamics and specificity of DNA tripler formation with isothermal titration calorimetry (ITC). The tripler formation between a 23-mer double-stranded homopurine-homopyrimidine and a 15-mer single-stranded homopyrimidine oligonucleotide forming T . AT and C+. GC triads at pH 4.8 is driven by a large negative calorimetric enthalpy change, Delta H-cal, of the order of -80 kcal/mol. Delta H-cal is strongly temperature dependent, yielding a heat capacity change, Delta C-p, of about -1 (kcal/molK-1. The equilibrium association constant, K, obtained from the titration curve is about 9 x 10(7) M(-1) at 25 degrees C (binding free energy change, Delta G, is about -11 kcal/mol). Thus, the tripler formation is accompanied by a negative entropy change (Delta S -245 (cal/molK-1 at 25 degrees C). We found that K is insensitive to temperature near room temperature, leading to an apparently small van't Hoff enthalpy change (Delta H-vH), in sharp contrast with the large negative Delta H-cal. Together, the analyses of the observed temperature dependences of K and Delta H and the large negative Delta C-p suggest that the tripler formation is a coupled process between conformational transitions in single-stranded DNA and its binding with double-stranded DNA. The examination of single mismatches in the tripler formation has shown that K and Delta G are not strongly affected by the particular combination of triad sequences (differences in Delta G are within 1.2 kcal/mol). In contrast, single mismatches affected Delta H-cal to a greater extent (up to 7-kcal/mol differences). We discuss possible means to enhance specificity in tripler formation, implied by the present findings.