To investigate potential structures of d(CGG/CCG)_n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), the structure of a single-stranded (ss) oligonucleotide containing d(CCG)₁₅ [ss(CCG)₁₅] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV absorbance, circular dichroism, chemical modification, and P1 nuclease digestion. ss(CCG)₁₅ had an unusually high pK_a (7.7 ± 0.2). At pH 8.5, ss(CCG)₁₅ formed a relatively unstable (T_m = 30 °C in 1 mM Na⁺) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C·⁺C base pairs, increased thermal stability (T_m = 37 °C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped away from the central portion of the helix. Examination of ss(CCG)₁₈ and ss(CCG)₂₀, which were designed to adopt hairpins containing alternative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pK_as of ∼8.2 and ∼8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)_n_≥₁₅ adopt hairpin conformations that contain CpG rather than GpC base-pair steps; the mismatched cytosines are protonated at physiological pH but are not H-bonded. We propose that protonation arises from the stacking of two cytosines in the minor groove of a distorted helix.

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