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The analytical methods for characterizing DNA sequence-dependent thermodynamic stability have been reviewed. A set of n-n sequence stability parameters is presented. Examples in which these values are used to calculate the thermodynamic stability of short duplex DNA oligomers are presented. The problem of determining sets of isothermal sequences is addressed by representing DNA sequences as graphs. Representing DNA sequences by a graph descriptor with special mathematical properties minimizes the computational difficulty of determining the number of DNA sequences with identical predicted thermodynamic stability. This is achieved by replacement of a whole set of sequences by a single representative. Applications of this concept were demonstrated for sequences assembled from individual bases and sequences assembled from oligomeric blocks.

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A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T(2)-biotin.dT-T(2) loop. The third base was a biotinylated uracil (U(B)) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3' dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3' end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5'-FITC, or radiolabeled with [gamma-(33)P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45 degrees C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin-target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

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Effects of different end sequences on stability, circular dichroism spectra (CD), and enzyme binding properties were investigated for six 22-base pair, non-self-complementary duplex DNA oligomers. The center sequences of these deoxyoligonucleotides have 8-14 base pairs in common and are flanked on both sides by sequences differing in context and A-T content. Temperature-induced melting transitions monitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for the six duplexes in buffered 115 mM Na(+) solutions. Values of the melting transition enthalpy, DeltaH(cal), and entropy, DeltaS(cal), were obtained directly from DSC experiments. Melting transition parameters, DeltaH(vH) and DeltaS(vH), were also estimated from van't Hoff analysis of optical melting curves collected as a function of DNA concentration, assuming a two-state melting transition. Melting free energies (20 degrees C) of the six DNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy as a function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the 22-base pair DNAs was also investigated. The central eight base pairs of all six molecules, 5'-A-GGATCC-A-3', contained a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concentration of each of the deoxyoligonucleotide substrates and analyzing reaction products by gel retardation. Binding isotherms of the total amount of bound DNA versus protein concentration were constructed which provided semiquantitative estimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers. Dissociation constants ranged from 0.5 x 10(-)(9) to 12.0 x 10(-)(9) M with corresponding binding free energies of -12.5 to -10.6 (+/-0. 1) kcal/mol. An inverse relationship is found when binding and stability are compared.

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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 A, or C, sequences (iota = 2, 3, 4, 6, 8, 10, 14). T 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 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 delta Gloop(n > iota) and single-strand circles, delta Gcir(N) as a function of end-loop size, tau = 2-14, circle size, N = 32 + 2 iota, 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 delta H, entropy delta S, and free energy delta G, 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 transition 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 iota loops (iota = 2, 3, 4, 6, 8, 10, 14).

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The DNA sequence of the chicken glutamine synthetase gene contains an A.T-rich stretch of approximately 1500 base pairs in the 3' noncoding regions of exon 7 [Pu, H., & Young, A. P. (1989) Gene 18, 169-175]. Within this region several palindromic sequences occur that could conceivably form intramolecular structures. One such perfect inverted repeat sequence resides between positions 2605 and 2623. To investigate the hairpin-forming potential for this sequence, optical and calorimetric melting and gel electrophoresis studies have been performed on the following synthetically prepared DNA and RNA oligomer subsequences: DNA, 5'd-T-T-T-T-T-T-A-A-T-A-A-T-T-A-A-A-A-A-A-3'; and RNA, 5'r-U-U-U-U-U-U-A-A-U-A-A-U-U-A-A-A-A-A-A-3'. The DNA strand corresponds to the coding strand sequence while the RNA strand represents the transcribed mRNA. Results of melting analysis of these 19-base, partially self-complementary strands performed in 115 mM Na+ yielded evaluations of their thermodynamic transition parameters. These values are consistent with the melting of unimolecular structures, presumably hairpins. Thermodynamic parameters evaluated by analysis of the optical melting transitions assuming a two-state model and measured directly by differential scanning calorimetry agreed within experimental error. Therefore, melting behavior of the hairpins is all-or-none like. The DNA hairpin is slightly more stable than the RNA hairpin with melting enthalpy delta H0 = 41.2 +/- 3.8 kcal/mol and entropy delta S0 = 133 +/- 11 cal/K.mol (eu) compared to delta H0 = 32.0 +/- 6.0 kcal/mol and entropy delta S0 = 105 +/- 20 eu for the RNA. Gel electrophoretic analysis of these oligomers alone and in various mixtures with their DNA and RNA complementary strands was also performed. Consistent with interpretations of melting results, these experiments revealed both strands alone preferentially form intramolecular hairpin structures. In mixtures in which their complementary strands are in vast molar excess (stoichiometric ratios > 10:1), the intramolecular structures are converted to intermolecular duplexes. For the DNA and RNA strands examined, the conversion is not complete until over a 1000-fold excess of the complementary strand is added. Semiquantitative analysis of gel electrophoretograms enabled evaluations of the relative free energies of the hairpin and duplex states as a function of complementary strand concentration. With the finding that these sequences preferentially form hairpins, potential roles these structures could play in regulatory activities are considered.

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Effects of different concentrations of Tetramethylammonium on the thermostability of six DNA dumbbells with similar well defined sequences have been investigated. Each molecule has a 16 base pair duplex stem linked on both ends by T4 single strand loops. Only the sequence of the four central base pairs distinguishes one molecule from the next. The distinguishing central sequences are, [A-T-A-T], [T-A-T-A], [A-A-A-A], [C-G-C-G], [G-C-G-C] and [G-G-G-G] situated between the sequences: 5'-G-T-A-T-C-C-[]-G-G-A-T-A-C-3' which are the same in all molecules. Optical melting curves collected on these molecules as a function of TMA concentration over the range from 0.09 M to 4.5 M revealed there is no single concentration of TMA where all these molecules exhibit the same melting temperature.

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