Nucleic Acids Research, 2002, Vol. 30, No. 17 3767-3777
© 2002 Oxford University Press
Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA
Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA
*To whom correspondence should be addressed. Tel: +1 816 235 5247; Fax: +1 816 235 1503; Email: thomasgj{at}umkc.edu
Present address:
Liviu Movileanu, Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA
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) [
HvHpm = 18.0 ± 1.6 kcal·mol–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (HvHpm = 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) (Gst = 0.8 ± 0.3 kcal· molbp–1), but marginal in poly(dA–dT)·poly(dA–dT) (Gst = 0.3 ± 0.3 kcal·molbp–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (HvHA = 407 ± 23 kcal·mol–1, SvHA = 1166 ± 67 cal·°K–1·mol–1, GvH(25°C)A = 60.0 ± 3.2 kcal·mol–1) are clearly distinguished from those of T (HvHT = 185 ± 38 kcal·mol–1, SvHT = 516 ± 109 cal·°K–1·mol–1, GvH(25°C)T = 27.1 ± 5.5 kcal·mol–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (HvHA = 333 ± 54 kcal·mol–1, SvHA = 961 ± 157 cal·°K–1·mol–1, GvH(25°C)A = 45.0 ± 7.6 kcal· mol–1; HvHT = 213 ± 30 kcal·mol–1, SvHT = 617 ± 86 cal·°K–1·mol–1, GvH(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.