Influence of pH on the equilibrium association constants for oligodeoxyribonucleotide-directed triple helix formation at single DNA sites.
The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrimidine.purine.pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 degrees C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3' (1) and 5'-TTTTTm5CTm5CTm5CTm5CTm5CT-3' (2, m5C is 2'-deoxy-5-methylcytidine) increased by 10- and 20-fold, respectively, from pH 7.6 to 5.8, indicating that the corresponding triple-helical complexes are stabilized by 1.4 and 1.7 kcal.mol-1, respectively, at the lower pH. Replacement of the five cytosine residues in 1 with 5-methylcytosine residues to yield 2 affords a stabilization of the triple helix by 0.1-0.4 kcal.mol-1 over the pH range 5.8-7.6. An analysis of these data in terms of a quantitative model for a general pH-dependent equilibrium transition revealed that pyrimidine oligonucleotides with cytidine and 5-methylcytidine form local triple-helical structures with apparent pKa's of 5.5 (C+GC triplets) and 5.7 (m5C+GC triplets), respectively, and that the oligonucleotides should bind to single sites on large DNA with apparent affinity constants of approximately 10(6) M-1 even above neutral pH.
Singleton
S F
SF
Arnold and Mabel Beckman Laboratories of Chemical Synthesis, California Institute of Technology, Pasadena 91125.
Dervan
P B
PB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D003596:Cytosine; D004247:DNA