At Physiological pH, d(CCG)15 Forms a Hairpin Containing Protonated Cytosines and a Distorted Helix

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Abstract
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 pKa (7.7 ± 0.2). At pH 8.5, ss(CCG)15 formed a relatively unstable (Tm = 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 (Tm = 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, pKas 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.