Thermodynamic, Spectroscopic, and Equilibrium Binding Studies of DNA Sequence Context Effects in Six 22-Base Pair Deoxyoligonucleotides

Abstract

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.

Influence of Buffer Species on the Thermodynamics of Short DNA Duplex Melting: Sodium Phosphate versus Sodium Cacodylate

“…Thermodynamic transition parameters ∆H°and ∆S°were determined from the normalized, baseline-corrected, ∆C p versus T curve via integration. 17,18 Analysis to obtain the calorimetric transition enthalpy (∆H°) and entropy (∆S°) was performed using the Synthetically prepared and purified single strands used to make the short oligoduplexes were synthesized on a one micromole scale and purchased from Integrated DNA Technologies (IDT). Average yield for each sample was 25-30 OD/ml.…”

Torsional Rigidities of Weakly Strained DNAs

“…These attempts all invoke the independent dinucleotide step model, wherein the secondary structure and torsional and bending rigidities associated with a given base-pair step are assumed to be completely independent of the sequence of its flanking DNA, or of any altered state of its flanking DNA that is induced by protein or other ligand binding, or by a B-to-Z transition. However, this assumption has been unequivocally contradicted by numerous published NMR structures of small duplexes, which yield different structural parameters for particular steps, e.g., A-A steps, embedded in different flanking sequences (46), as well as by numerous other published experiments (6,36,(47)(48)(49)(50)(51)(52) and unpublished studies of S. A. Winkle (Florida International University, personal communication, 1997) that were reviewed previously (36). Other evidence indicates that dinucleotide step models for directional permanent bends, or for A d , cannot account satisfactorily for the behavior of all sequences (43,53,54).…”

Applications of Biocalorimetry: Binding, Stability and Enzyme Kinetics

“…[100][101][102] This pioneering work was limited to examining a small number of synthetic polymers and DNA samples purified from biological material. [104][105][106] Often the focus of these studies has been to examine sequence-dependent effects on the thermodynamic stability of DNA in the context of nearest-neighbour base pair interactions. However, despite these limitations the thermodynamic data obtained by these workers have been subsequently confirmed and validated as DSC technology has advanced and the availability of purified nucleic acids has increased.…”