Thermodynamic, Spectroscopic, and Equilibrium Binding Studies of DNA Sequence Context Effects in Six 22-Base Pair Deoxyoligonucleotides
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- 作者：Peter V. Riccelli ; Peter M. Vallone ; Irina Kashin ; Brian D. Faldasz ; Michael J. Lane ; and Albert S. Benight
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 24, 1999
- 年：1999
- 卷：38
- 期：34
- 页码：11197 - 11208
- 全文大小：155K
- 年卷期：v.38,no.34(August 24, 1999)
- ISSN：1520-4995
文摘
Effects of different end sequences on stability, circular dichroism spectra (CD), and enzymebinding 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 flankedon both sides by sequences differing in context and A-T content. Temperature-induced melting transitionsmonitored by differential scanning calorimetry (DSC) and ultraviolet absorbance were measured for thesix duplexes in buffered 115 mM Na+ solutions. Values of the melting transition enthalpy, 
mages/gifchars/Delta.gif" BORDER=0 >Hcal, andentropy, mages/gifchars/Delta.gif" BORDER=0 >Scal, were obtained directly from DSC experiments. Melting transition parameters, mages/gifchars/Delta.gif" BORDER=0 >HvH andmages/gifchars/Delta.gif" BORDER=0 >SvH, were also estimated from van't Hoff analysis of optical melting curves collected as a function ofDNA concentration, assuming a two-state melting transition. Melting free energies (20 mages/entities/deg.gif">C) of the sixDNAs evaluated from DSC experiments ranged from -18.7 to -32.7 kcal/mol. van't Hoff estimates ofthe free energies ranged from -18.5 to -48.0 kcal/mol. With either method, the trends in free energy asa function of sequence were identical. Equilibrium binding by BamHI restriction endonuclease to the22-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 bindingassays 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 totalamount of bound DNA versus protein concentration were constructed which provided semiquantitativeestimates of the equilibrium dissociation constants for dissociation of BamHI from the six DNA oligomers.Dissociation constants ranged from 0.5 × 10-9 to 12.0 × 10-9 M with corresponding binding free energiesof -12.5 to -10.6 (±0.1) kcal/mol. An inverse relationship is found when binding and stability arecompared.