AT PHYSIOLOGICAL PH, D(CCG)(15) FORMS A HAIRPIN CONTAINING PROTONATEDCYTOSINES AND A DISTORTED HELIX

To investigate potential structures of d(CGG/CCG)(n) that might relate to their biological function and association with tripler repeat expa nsion diseases (TREDs), the structure of a single-stranded (ss) oligon ucleotide containing d(CCG)(15) [ss(CCG)(15)] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV a bsorbance, circular dichroism, chemical modification, and P1 nuclease digestion. ss(CCG)(15) had an unusually high pK(a) (7.7 +/- 0.2). At p H 8.5, ss(CCG)(15) formed a relatively unstable (T-m = 30 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hair pin contained protonated cytosines but no detectable C .C+ base pairs, increased thermal stability (T-m = 37 degrees C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped awa y from the central portion of the helix. Examination of ss(CCG)(18) an d ss(CCG)(20), which were designed to adopt hairpins containing altern ative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pK(a)s of similar to 8.2 and similar to 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing ( CCG)(n greater than or equal to 15) adopt hairpin conformations that c ontain CpG rather than GpC base-pair steps; the mismatched cytosines a re protonated at physiological pH but are not H-bonded. We propose tha t protonation arises from the stacking of two cytosines in the minor g roove of a distorted helix.