Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in six 22-base pair deoxyoligonucleotides

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 thes e deoxyoligonucleotides have 8-14 base pairs in common and are flanked on b oth sides by sequences differing in context and A-T content. Temperature-in duced melting transitions monitored by differential scanning calorimetry (D SC) and ultraviolet absorbance were measured for the six duplexes in buffer ed 115 mM Na+ solutions. Values of the melting transition enthalpy, Delta H -cal, and entropy, Delta S-cal, were obtained directly from DSC experiments . Melting transition parameters, Delta H-vH and Delta S-vH, were also estim ated from van't Hoff analysis of optical melting curves collected as a func tion of DNA concentration, assuming a two-state melting transition. Melting free energies (20 OC) of the six DNAs evaluated from DSC experiments range d from -18.7 to -32.7 kcal/mol. van't Hoff estimates of the free energies r anged 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 Bam HI 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', conta ined a BamHI recognition sequence bounded by A-T base pairs. Magnesium free binding assays were performed by titering BamHI against a constant concent ration of each of the deoxyoligonucleotide substrates and analyzing reactio n products by gel retardation. Binding isotherms of the total amount of bou nd DNA versus protein concentration were constructed which provided semiqua ntitative estimates of the equilibrium dissociation constants for dissociat ion 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 o f -12.5 to -10.6 (+/- 0.1) kcal/mol. An inverse relationship is found when binding and stability are compared.