At Physiological pH, d(CCG)15 Forms a Hairpin Containing Protonated Cytosines and a Distorted Helix
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- 作者：Adong Yu ; Maria D. Barron ; Rebecca M. Romero ; Mellisa Christy ; Barry Gold ; Jianli Dai ; Donald M. Gray ; Ian S. Haworth ; and Michael Mitas
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：March 25, 1997
- 年：1997
- 卷：36
- 期：12
- 页码：3687 - 3699
- 全文大小：345K
- 年卷期：v.36,no.12(March 25, 1997)
- ISSN：1520-4995
文摘
To investigate potential structures ofd(CGG/CCG)n that might relate to theirbiological functionand association with triplet repeat expansion diseases (TREDs), thestructure of a single-stranded (ss)oligonucleotide containing d(CCG)15[ss(CCG)15] was examined by studies of the pH andtemperaturedependence of electrophoretic mobility, UV absorbance, circulardichroism, chemical modification, andP1 nuclease digestion. ss(CCG)15 had an unusuallyhigh pKa (7.7 ± 0.2). At pH 8.5,ss(CCG)15 formeda relatively unstable (Tm = 30 
C in 1 mMNa+) hairpin containing CpG base-pair steps. At pH7.5, thehairpin contained protonated cytosines but no detectableC·+C base pairs, increased thermal stability(Tm= 37 C), increased stacking of the CpG base-pair steps, and asingle cytosine that was flipped awayfrom the central portion of the helix. Examination ofss(CCG)18 and ss(CCG)20, which weredesigned toadopt hairpins containing alternative GpC base-pair steps, revealedhairpins containing CpG base-pairsteps, pKas of ~8.2 and ~8.4, respectively,and distorted helices. The results suggest that DNAsequencescontaining (CCG)n
15adopt hairpin conformations that contain CpG rather than GpC base-pairsteps; themismatched cytosines are protonated at physiological pH but are notH-bonded. We propose thatprotonation arises from the stacking of two cytosines in the minorgroove of a distorted helix.
C in 1 mMNa+) hairpin containing CpG base-pair steps. At pH7.5, thehairpin contained protonated cytosines but no detectableC·+C base pairs, increased thermal stability(Tm= 37 C), increased stacking of the CpG base-pair steps, and asingle cytosine that was flipped awayfrom the central portion of the helix. Examination ofss(CCG)18 and ss(CCG)20, which weredesigned toadopt hairpins containing alternative GpC base-pair steps, revealedhairpins containing CpG base-pairsteps, pKas of ~8.2 and ~8.4, respectively,and distorted helices. The results suggest that DNAsequencescontaining (CCG)n15adopt hairpin conformations that contain CpG rather than GpC base-pairsteps; themismatched cytosines are protonated at physiological pH but are notH-bonded. We propose thatprotonation arises from the stacking of two cytosines in the minorgroove of a distorted helix.