The energetics of oligodeoxyribonucleotide-directed triple helix formation for the pyrim-idine-purine-pyrimidine structural motif were determined over the pH range 5.8-7.6 at 22 C (100 mM Na+ and 1 mM spermine) using quantitative affinity cleavage titration. The equilibrium binding constants for 5'-TTTTTCTCTCTCTCT-3'(1) and S'-TTTTTm^ Tm^ Tm^ Tm^ Tn^ CT-S'(2, m5Cis 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~ 106 M'1 even above neutral pH.

Oligonucleotide-directed triple helix formation provides a versatile structural motif for the design of molecules capable of sequence-specific recognition of double-helical DNA (Moser & Dervan, 1987; LeDoan et al., 1987; Cooney et al., 1988). Pyrimidine oligodeoxyribonucleotides bind in the major groove of double-helical DNA parallel to a purine-rich strand of the Watson-Crick duplex to form a local pyrimidine-purinepyrimidine triple-helical structure (Moser & Dervan, 1987; Praseuth et al., 1988; de los Santos et al., 1989; Rajagopal & Feigon, 1989a, b). The sequence specificity for the binding of an oligonucleotide to its double-helical target site is achieved