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 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.
Riccelli
P V
PV
Department of Chemistry, University of Illinois, Chicago 60607, USA.
Vallone
P M
PM
Kashin
I
I
Faldasz
B D
BD
Lane
M J
MJ
Benight
A S
AS
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D004247:DNA; D015245:Deoxyribonuclease BamHI