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) [ΔH(vH)(pm) = 18.0 ± 1.6 kcal·mol(–1) (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH(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), ΔS(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.