ALBERT

Notes: Seventeen DNA dumbbells were constructed that have duplex sequences ranging in length from 14 to 18 base pairs linked on the ends by T4 single-strand loops. Fifteen of the molecules have the core duplexes with the sequences 5′G-T-A-T-C-C-(W-X-Y-Z)-G-G-A-T-A-C3′, where (W-X-Y-Z) represents a unique combination of A · T, T · A, G · C, and C · G base pairs. The remaining two molecules have the central sequences (W-X-Y-Z) = A-C and A-C-A-C-A-C. These duplex sequences were designed such that the central sequences include different combinations of the 10 possible nearest-neighbor (n-n) stacks in DNA. In this sense the set of molecules is complete and serves as a model system for evaluating sequence-dependent local stability of DNA. Optical melting curves of the samples were collected in 25, 55, 85, and 115 mM [Na +], and showed, regardless of solvent ionic strength, that the transition temperatures of the dumbbells vary by as much as 14° for different molecules of the set.Results of melting experiments analyzed in terms of a n-n sequence-dependent model allowed evaluation of nine independent linear combinations of the n-n stacking interactions in DNA as a function of solvent ionic strength. Although there are in principle 10 possible different n-n interactions in DNA, these 10 are not linearly independent and therefore can not be uniquely determined. For molecules with ends, there are 9 linearly independent combinations, as opposed to circular or semiinfinite repeating copolymers where only 8 linear combinations of the 10 possible n-n interactions are linearly independent. The n-n interactions are presented as combinations of the deviations from average stacking for the 5′-3′ base-pair doublets, δGi, and reveal several interesting features: (1) Titratable changes in the values of δGi, with changing salt environment are observed. In all salts the most stable unique combination is δG4 = (δGGpC + δGCpG,)/2, and the least stable is the GpG/CpC stack, δG2 = δGGpG/CpC. (2) The χ2 values of the fits of the evaluated δGi's to experimental data increased with decreasing [Na +], suggesting that significant interactions beyond nearest neighbors become more pronounced, particularly at 25 mM Na +. (3) In 85 and 115 mM Na +, where the n-n approximation seems to be most valid, the absolute value of δGi for any n-n stack or average of two n-n stacks is not more than ∼ 220 cal/mole, indicating that deviations from average stacking due to n-n interactions represent about 15% of the total stability of a base pair. The overall thermodynamic stability of DNA is predominantly determined by the sequence content (%G · C). Even though the contribution of n-n interactions to overall stability are intrinsically small, reliable predictions of DNA transition temperatures de novo from sequence can be significantly compromised by cumulative errors in the δGi's. (4) Comparisons of our set of n-n linear combinations evaluated in 115 mM Na+ with various published sets evaluated from melting experiments of long restriction fragments, synthetic polymers, and short oligomers, and those obtained from a reanalysis of published melting data of synthetic polymers, are presented. The analysis reveals a major consensus agreement between n-n free energies evaluated from melting data of restriction fragments and long synthetic repeating copolymers. In contrast, only a minor consensus agreement is obtained between our n-n set and these values or those obtained from melting analysis of a combination of short oligomers and long polymers or those theoretically calculated. Results of these comparisons suggest the values of n-n interactions evaluated from DNA melting curves depend on the length of the melted duplex regions of the DNA molecules that comprise the sample set.

Notes: The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C12H24 (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T4 loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na+ and revealed, regardless of [Na +], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T4 loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T4 end loops are essentially two state in 25 and 55 mM Na+. In contrast, significant deviations from two-state behavior were observed in 85 m MNa+. From our previously published melting data of DNA dumbbells with Tn end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881-892] and the dumbbell with T4 end loops of this study, a plot of d(Tm)/d ln [Na+] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (Tm)/d ln [Na+] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.

Notes: Optical melting transitions of the short DNA hairpins formed from the self-complementary DNA oligomers d[GGATACX4GTATCC] where X = A, T, G, or C measured in 100 mM NaCl are presented. A significant dependence of the melting transitions on loop sequence is observed and transition temperatures, tm, of the hairpins vary from 58.3°C for the T4 loop hairpin to 55.3°C for the A4 loop. A nearest-neighbor sequence-dependent theoretical algorithm for calculating melting curves of DNA hairpins is presented and employed to analyze the experimental melting transitions. Experimental melting curves were fit by adjustment of a single theoretical parameter, Fend(n), the weighting function for a hairpin loop comprised of n single-strand bases. Empirically determined values of Fend(n) provide an evaluation of the free-energy of hairpin loop formation and stability. Effects of heterogeneous nearest-neighbor sequence interactions in the duplex stem on hairpin loop for mation were investigated by evaluating Fend(n) in individual fitting procedures using two of the published sets of nearest-neighbor stacking interactions in DNA evaluated in 100 mM NaCl and given by Wartell and Benight, 1985. In all cases, evaluated values of Fend(n) were obtained that provided exact theoretical predictions of the experimental transitions.Results of the evaluations indicate: (1) Evaluated free-energies of hairpin loop formation are only slightly dependent on loop sequences examined. At the transition temperature, Tm, the free-energy of forming a loop of four bases is approximately equal for T4, G4, or C4 loops and varies from 3.9 to 4.8 kcal/mole depending on the set of nearest-neighbor interactions employed in the evaluations. This result suggests, in light of the observed differences in stability between the T4, G4, and C4 loop hairpins, that sequence-dependent interactions between base residues of the loop are most likely not the source of the enhanced stability of a T4 loop. In contrast, the evaluated free-energy of forming an A4 loop is approximately 400 cal/mole higher for each nearest-neighbor set indicating unfavorable interactions between A bases in a loop-affect loop formation and overall hairpin stability, (2) The absolute value for the free-energy of loop formation at the Tm of each hairpin varies by about 1 kcal/mole depending on the set of nearest-neighbor interactions employed and the relative hierarchy of stability for each loop is conserved for different nearest neighbor sets, (3) The melting process of each hairpin deviates from strict two-state behavior in the order according to loop sequence of T 〉 A 〉 G 〉 C, (4) Results of our analysis are compared with the early work of Scheffler et al., 1970 on the hairpins formed from the copolymer sequences d(T - A)q where q = 9-21. Comparisons with the more recent works a DNA dumbbell (Benight et al., 1988) and the very similar DNA hairpins studied by Senior et al., 1988 are presented.

Notes: 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.

Notes: Optical melting curves of seven DNA dumbbells with the 16 base-pair duplex sequence 5′G-C-A-T-A-G-A-T-G-A-G-A-A-T-G-C3′ linked on both ends by Tn (n = 2, 3, 4, 6, 8, 10, and 14) loops measured in 30, 70, and 120 mM Na+ are analyzed in terms of the numerically exact statistical thermodynamic model of DNA melting. The construction and characterization of these molecules were described in the previous paper (Amaratunga et al., 1992). As was recently reported for hairpins (T. M. Paner, M. Amaratunga, M. J. Doktycz, and A. S. Benight, 1990, Biopolymers, Vol. 29, pp. 1715-1734) theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting the parameters representing loop and circle formation to optimize the fits. The systematically determined empirical parameters provide evaluations of the free energies of hairpin loop formation ΔGloop(n) and single-strand circles ΔGcircle(N), as a function of end loop size, n = 2-14, and circle size, N = 32 + 2n. The dependence of these quantities on solvent ionic strength over the range from 30 to 120 mM Na+ was evaluated. An approximately analytical expression for the partition function Q(T) of the dumbbells was formulated that allowed a means for determining the transition enthalpy ΔH° and entropy ΔS° for every dumbbell, revealing the dependence of these quantities on loop size. In this multistate approach a manifold of partially melted intermediate microstates are considered and therefore no assumptions regarding the nature of the melting transitions (that they are two-state) are required. The transition thermodynamic parameters were also determined from a van't Hoff analysis of the melting curves. Comparisons between the results of the multistate analysis and the two-state van't Hoff analysis revealed significant differences for the dumbbells with larger end loops, indicating that the melting transitions of the larger looped dumbbells deviate considerably from two-state behavior. Results are then compared with published melting studies of much larger DNA dumbbells (D. B. Naritsin and Y. L. Lyubchenko, 1990, Journal of Biomolecular Structure and Dynamics, Vol. 8, pp. 1-13), of small hairpins (Paner et al., 1990; M. J. Doktycz, T. M. Paner, M. Amaratunga and A. S. Benight, 1990, Biopolymers, Vol. 30, pp. 829-845) and another dumbbell (A. S. Benight, J. M. Schurr, P. F. Flynn, B. R. Reid, and D. E. Wemmer, 1988) Journal of Molecular Biology, Vol. 200, (pp. 377-399). ΔGloop (n = 4) and ΔGcircle (40) were also evaluated from analysis of the melting curves of the 15 DNA dumbbells with 16 base-pair stems presented in the first paper (Doktycz et al., 1992). With the exception of the dumbbell with the central sequence 5′-T-T-A-A-3′, these quantities were found to be virtually independent of the sequence identity of the central 4 base pairs of the stem.

Notes: CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ∼20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying.On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (∼ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of fT(T), the fraction of triplex as a function of temperature, was obtained. Analysis of fT vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔHT = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔST = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from fT = 0 to 0.55 and a higher, sharper transition above fT = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′T = 44.8 ± 9.6 kcal/mol and ΔS′T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′T = 128.0 ± 2.3 kcal/mol and ΔS′T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.

Notes: Optical melting curves of 22 DNA dumbbells with the 16-base pair duplex sequence 5′-G-C-A-T-C-A-T-C-G-A-T-G-A-T-G-C-3′ linked on both ends by single-strand loops of At or Ct sequences (˛ = 2, 3, 4, 6, 8, 10, 14), Tt sequences (˛ = 2, 3, 4, 6, 8, 10), and Gt sequences (t = 2, 4) were measured in phosphate buffered solvents containing 30, 70, and 120 mM 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 three base nucleotide loops. Experimental melting curves were analyzed in terms of the numerically exact (multistate) statistical thermodynamic model of DNA dumbbell melting previously described (T. M. Paner, M. Amaratunga & A. S. Benight (1992), Biopolymers 32, 881). Theoretically calculated melting curves were fitted to experimental curves by simultaneously adjusting model parameters representing statistical weights of intramolecular hairpin loop and single-strand circle states. The systematically determined empirical parameters provided evaluations of the energetics of hairpin loop formation as a function of loop size, sequence, and salt environment. Values of the free energies of hairpin loop formation ΔGloop(n 〉 t) and single-strand circles, ΔGcir(N) as a function of end-loop size, t = 2-14, circle size, N = 32 + 2t, and loop sequence were obtained. These quantities were found to depend on end-loop size but not loop sequence. Their empirically determined values also varied with solvent ionic strength. Analytical expressions 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 ΔH, entropy ΔS, and free energy ΔG, were evaluated for the dumbbells as a function of end-loop size, sequence, and [Na+]. Since the multistate analysis is based on the numerically exact model, and considers a statistically significant number of theoretically possible partially 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, thermodynamic transition parameters were also evaluated directly from experimental melting curves assuming a two-state transition and using the graphical van't Hoff analysis. Comparisons between results of the multistate 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 deviate from two-state behavior.Dependence of thermodynamic parameters on[Na+] as a function of loop size suggests single-strand end-loops have different counterion binding properties than the melted circle. Results are compared with those obtained in an earlier study of dumbbells with the slightly 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 loops (˛ = 2,3,4,6,8,10,14).© 1996 John Wiley &Sons, Inc.

Notes: Many important applications of DNA sequence-dependent hybridization reactions have recently emerged. This has sparked a renewed interest in analytical calculations of sequence-dependent melting stability of duplex DNA. In particular, for many applications it is often desirable to accurately predict the transition temperature, or tm, of short duplex DNA oligomers (∼ 20 base pairs or less) from their sequence and concentration. The thermodynamic analytical method underlying these predictive calculations is based on the nearest-neighbor model. At least 11 sets of nearest-neighbor sequence-dependent thermodynamic parameters for DNA have been published. These sets are compared. Use of the nearest-neighbor sets in predicting tm from the DNA sequence is demonstrated, and the ability of the nearest-neighbor parameters to provide accurate predictions of experimental tm's of short duplex DNA oligomers is assessed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 217-239, 1997