STUDIES OF DNA DUMBBELLS .6. ANALYSIS OF OPTICAL MELTING CURVES OF DUMBBELLS WITH A 16-BASE PAIR DUPLEX STEM AND END-LOOPS OF VARIABLE SIZEAND SEQUENCE

Optical melting curves of 22 DNA dumbbells with the 16-base pair duple x sequence 5'-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3' linked on both ends b y single-strand loops of A(iota) or C-iota sequences (iota = 2, 3, 4, 6, 8, 10, 14), T-iota sequences (iota = 2, 3, 4, 6, 8, 10), and G(iota ) sequences (iota = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 nM Na+. For dumbbells with loops comprised of at least three nucleotides, stability is inversely proportional to end-loop size. Dumbbells with loops comprised of only two nucleotide bases generally have lower stabilities than dumbbells with loops compr ised of only two nucleotide bases generally have lower stabilities tha n dumbbells with three base nucleotide loops. Experimental melting cur ves were analyzed in terms of the numerically exact (multistate) stati stical thermodynamic model of DNA dumbbell melting previously describe d (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimen tal curves by simultaneously adjusting model parameters representing s tatistical weights of intramolecular hairpin loop and single-strand ci rcle states. The systematically determined empirical parameters provid ed evaluations of the energetics of hairpin loop formation as a functi on of loop size, sequence, and salt environment. Values of the free en ergies of hairpin loop formation Delta G(loop)(n > iota) and single-st rand circles, Delta G(cir)(N) as a function of end-loop size, iota = 2 -14, circle size, N = 32 + 2 iota, and loop sequence were obtained. Th ese quantities were found to depend on end-loop size but not loop sequ ence. Their empirically determined values also varied with solvent ion ic strength. Analytical expression for the partition function Q(T) of the dumbbells were evaluated using the empirically evaluated best-fit loop parameters. From Q(T), the melting transition enthalpy Delta H, e ntropy Delta S, and free energy Delta G, were evaluated for the dumbbe lls as a function of end-loop size, sequence, and [Na+]. Since the mul tistate analysis is based on the numerically exact model, and consider s a statistically significant number of theoretically possible partial ly melted states, it does not require prior assumptions regarding the nature of the melting transition, i.e., whether or not it occurs in a two-state manner. For comparison with the multistate analysis, thermod ynamic transition parameters were also evaluated directly from experim ental melting curves assuming a two-state transition and using the gra phical van't Hoff analysis. Comparisons between results of the multist ate and two-state analyses suggested dumbbells with loops comprised of six or fewer residues melted in a two-state manner, while the melting processes for dumbbells with larger end-loops derivate from two-state behavior. Dependence of thermodynamic transitions parameters on [Na+] as a function of loop size suggests single-strand end-loops have diff erent counterion binding properties than the melted circle. Results ar e compared with those obtained in an earlier study of dumbbells with s lightly different stem sequence 5'-G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C-3' linked on the ends by t(iota) loops(iota = 2, 3, 4, 6, 8, 10, 14). (C) 1996 John Wiley & Sons, Inc.