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 fo rmation between a 23-mer double-stranded homopurine-homopyrimidine and a 15-mer single-stranded homopyrimidine oligonucleotide forming T . A T and C+. GC triads at pH 4.8 is driven by a large negative calorimetr ic enthalpy change, Delta H-cal, of the order of -80 kcal/mol. Delta H -cal is strongly temperature dependent, yielding a heat capacity chang e, Delta C-p, of about -1 (kcal/molK-1. The equilibrium association co nstant, 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 kca l/mol). Thus, the tripler formation is accompanied by a negative entro py 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 app arently small van't Hoff enthalpy change (Delta H-vH), in sharp contra st with the large negative Delta H-cal. Together, the analyses of the observed temperature dependences of K and Delta H and the large negati ve Delta C-p suggest that the tripler formation is a coupled process b etween conformational transitions in single-stranded DNA and its bindi ng with double-stranded DNA. The examination of single mismatches in t he tripler formation has shown that K and Delta G are not strongly aff ected by the particular combination of triad sequences (differences in Delta G are within 1.2 kcal/mol). In contrast, single mismatches affe cted Delta H-cal to a greater extent (up to 7-kcal/mol differences). W e discuss possible means to enhance specificity in tripler formation, implied by the present findings.