Thermodynamic stability of the 5′ dangling‐ended DNA hairpins formed from sequences 5′‐(XY)₂GGATAC(T)₄GTATCC‐3′, where X, Y = A,T,G,C

Expressions for the partition function Q(T) of DNA hairpins are presented. Calculations of Q(T), in conjunction with our previously reported numerically exact algorithm [T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight (1990) Biopolymers, 29, 1715–1734], yield a numerical method to evaluate the temperature dependence of the transition enthalpy, entropy, and free energy of a DNA hairpin directly from its optical melting curve. No prior assumptions that the short hairpins melt in a two‐state manner are required. This method is then applied in a systematic manner to investigate the stability of the six base‐pair duplex stem 5′‐GGATAC‐3′ having four‐base dangling single‐strand ends with the sequences (XY)₂, where X, Y = A, T, G, C, on the 5′ end and a T⁴ loop on the 3′ end. Results show that all dangling ends of the sample set stabilize the hairpin against melting. Increases in transition temperatures as great as 4.0°C above the blunt‐ended control hairpin were observed. The hierarchy of the hairpin transition temperatures is dictated by the identity of the first base of the dangling end adjoining the duplex in the order: purine > T > C. Calculated melting curves of every hairpin were fit to experimental curves by adjustment of a single parameter in the numerically exact theoretical algorithm. Exact fits were obtained in all cases. Experimental melting curves were also calculated assuming a two‐state melting process. Equally accurate fits of all dangling‐ended hairpin melting curves were obtained with the two‐state model calculation. This was not the case for the melting curve of the blunt‐ended hairpin, indicating the presence of a four‐base dangling‐end drives hairpin melting to a two‐state process. Q(T) was calculated as a function of temperature for each hairpin using the theoretical parameters that provided calculated curves in exact agreement with the experimentally obtained optical melting curves. From Q(T), the temperature dependence of the transition enthply ΔH, enytropy ΔS, and free energy ΔG were calculated for every hairpin providing a quantitative assessment of the effects of dangling ends on hairpin thermodynamics. Comparisons of our results are made with those of the Breslauer group [M. Senior, R. A. Jones, and K. J. Breslauer (1988) Biochemistry 27, 3879–3885] on the T₂5′ dangling‐ended d (GC)₃ duplexes. To estimate the average contribution to stability of each single‐stand nearest neighbor stack to the duplex stem, the relative values ΔΔH and ΔΔS of the transition enthalpy and entropy for each dangling‐ended hairpin compared to the blunt‐ended control hairpin were cast in a system of 16 equation in the 16 unkowns, The nonsingular system of equations was solved for the unknowns by matrix diagonalization, which yielded the relative average contribution of each of the 16 possible nearest neighbor dinucleotide 5′3′ stacks in single strand DNA to the stability of a 5′‐GGATAC‐3′ duplex stem.