Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

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) [Δ_vH^pm = 18-0 ± 1.6 kcal·mol^-1 (kilocalories per mole cooperative unit)] and poly(dA-dT)·poly(dA-dT) (Δ_vH^pm = 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·mol_bp^-1 (kilocalories per mole base pair)] is greater than that of poly(dA-dT). poly(dA-dT) (-6.31 kcal·mol_bp^-1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^-1), but marginal in poly (dA-dT)·poly(dA-dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^-1). (v) In poly(dA)·poly(dT), the van't Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^-1, ΔS_vH^A = 1166 ± 67 cal·°K^-1·mol^-1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^-1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^-1, ΔS_vH^T = 516 ± 109 cal·°K^-1·mol^-1, ΔG_vH(25°C₎^T = 27.1 ± 5.5 kcal·mol^-1). (vi) Similar relative differences are observed in poly(dA-dT)·poly(dA-dT) (ΔH_vH^A = 333 ± 54 kcal·mol^-1, ΔS_vH^A = 961 ± 157 cal·°K^-1·mol^-1, ΔG_vH(25°_C)^A = 45.0 ± 7.6 kcal·mol^-1; ΔH_vH^T = 213 ± 30 kcal·mol^-1, Δ_vH^T 617 ± 86 cal·°K^-1*mol^-1, ΔG_vH(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.