At physiological pH, d(CCG)15 forms a hairpin containing protonated cytosines and a distorted helix.
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 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C x +C base pairs, increased thermal stability (Tm = 37 degrees 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 approximately 8.2 and approximately 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)(n > or = 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.
Yu
A
A
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-3035, USA.
Barron
M D
MD
Romero
R M
RM
Christy
M
M
Gold
B
B
Dai
J
J
Gray
D M
DM
Haworth
I S
IS
Mitas
M
M
D001483:Base Sequence; D003596:Cytosine; D004247:DNA; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D018911:Trinucleotide Repeats
D006898:Hydroxylamines; D019811:Hydroxylamine; D003596:Cytosine; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases