Thermodynamic, spectroscopic, and equilibrium binding studies of DNA sequence context effects in four 40 base pair deoxyoligonucleotides

Effects of different end sequences on melting, circular dichroism spectra ( CD), and enzyme binding properties were investigated for four 40 base pair, non-self-complementary duplex DNA oligomers. The center sequences of these oligoduplexes have either of two 22 base pair modules flanked on both side s by sequences differing in AT content. Temperature-induced melting transit ions monitored by differential scanning calorimetry (DSC) and ultraviolet a bsorbance were measured for the six duplexes in buffered 115 mM Na+ solutio ns. Values of the melting transition enthalpy, Delta H-cal, and entropy, De lta S-cal, were obtained directly from DSC experiments. Melting transition parameters, Delta H-vH and Delta S-vH, were also estimated from a van't Hof f analysis of optical melting curves collected as a function of DNA concent ration, assuming that the melting transition is two-state. Melting free ene rgies (20 degrees C) evaluated from DSC melting experiments on the four dup lex DNAs ranged from -52.2 to -77.5 kcal/mol. Free energies based on the va n't Hoff analysis were -37.9 to -58.8 kcal/mol. Although the values are dif ferent, trends in the melting free energies of the four duplex DNAs as a fu nction of sequence were identical in both DSC and optical analyses. Subject to several assumptions, values for the initiation free energy were estimat ed for each duplex, defined as Delta G(int) = Delta G(cal) - Delta G(pred), where Delta G(cal) is the experimental free energy at 20 degrees C determi ned from the experimentially measured values of the transition enthalpy, De lta H-cal, and entropy, Delta S-cal. The predicted free energy of the seque nce, Delta G(pred)(20 degrees C), is obtained using published nearest-neigh bor sequence stability values. For three of the four duplexes, values of De lta G(int) are essentially nil. In contrast, the duplex with 81.8% GC has a considerably higher estimate of Delta G(int) = 7.1 kcal/mol. The CD spectr a for the six duplexes collected over the wavelength range from 200 to 320 nm are also sequence-dependent, Factor analysis of the CD spectra by singul ar value decomposition revealed that the experimental CD spectra could be r econstructed from linear combinations of two minor and one major subspectra . Changes in the coefficients of the major subspectrum for different sequen ces reflect incremental sequence-dependent variations of the CD spectra. Eq uilibrium binding by BamHI restriction endonuclease to the 40 base pair DNA s whose central eight base pairs contain the recognition sequence for BamHI restriction enzyme bounded by A . T base pairs, 5'-A-GGATCC-A-3' was inves tigated. Binding assays were performed by titering BamHI against a constant concentration of each of the duplex DNA substrates, in the absence of Mg2, followed by analysis by gel retardation. Under the conditions employed, t he enzyme binds but does not cleave the DNAs. Results of the assays reveale d two binding modes with retarded gel mobilities. Binding isotherms for the fraction of bound DNA species versus enzyme concentration for each binding mode were constructed and analyzed with a simple two-step equilibrium bind ing model. This analysis provided semiquantitative estimates on the equilib rium binding constants for BamHI to the four DNAs. Values obtained for the binding constants varied only 7-fold and ranged from 6 x 10(-8) to 42 x 10( -8) M, with binding free energies from -8.6 to -9.7 (+/- 0.2) kcal/mol depe nding on the sequence that flanks the enzyme binding site. Unlike what was found earlier in binding studies of the 22 base pair duplex es that constitute the core modules of the present 40-mers [Riccelli, P. V. , Vallone, P. M., Kashin, I., Faldasz, B. D., Lane, M. J., and Benight, A. S. (1999) Biochemistry 38, 11197-11208], no obvious relationship between bi nding and stability was found for these longer DNAs. Apparently, effects of flanking sequence stability on restriction enzyme binding may only be meas urable in very short duplex deoxyoligonucleoties.