In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA-dT)·poly(dA-dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA-dT)·poly(dA-dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van't Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔvHpm = 18-0 ± 1.6 kcal·mol-1 (kilocalories per mole cooperative unit)] and poly(dA-dT)·poly(dA-dT) (ΔvHpm = 13.4 ± 2.5 kcal·mol-1) differ by an amount (∼4.6 kcal·mol-1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)n·(dT)n tracts. (iii) The overall stacking free energy of poly(dA)·poly(dT) [-6.88 kcal·molbp-1 (kilocalories per mole base pair)] is greater than that of poly(dA-dT). poly(dA-dT) (-6.31 kcal·molbp-1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔGst = 0.8 ± 0.3 kcal· molbp-1), but marginal in poly (dA-dT)·poly(dA-dT) (ΔΔGst = 0.3 ± 0.3 kcal·molbp-1). (v) In poly(dA)·poly(dT), the van't Hoff parameters for melting of A (ΔHvHA = 407 ± 23 kcal·mol-1, ΔSvHA = 1166 ± 67 cal·°K-1·mol-1, ΔGvH(25°C)A = 60.0 ± 3.2 kcal·mol-1) are clearly distinguished from those of T (ΔHvHT = 185 ± 38 kcal·mol-1, ΔSvHT = 516 ± 109 cal·°K-1·mol-1, ΔGvH(25°C)T = 27.1 ± 5.5 kcal·mol-1). (vi) Similar relative differences are observed in poly(dA-dT)·poly(dA-dT) (ΔHvHA = 333 ± 54 kcal·mol-1, ΔSvHA = 961 ± 157 cal·°K-1·mol-1, ΔGvH(25°C)A = 45.0 ± 7.6 kcal·mol-1; ΔHvHT = 213 ± 30 kcal·mol-1, ΔvHT 617 ± 86 cal·°K-1*mol-1, ΔGvH(25°C)T = 29.3 ± 4.9 kcal·mol-1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.
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