Unfolding Kinetics of the Human Telomere i-Motif Under a 10 pN Force Imposed by the 伪-Hemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH

详细信息    查看全文

• 作者：Yun Ding ; Aaron M. Fleming ; Lidong He ; Cynthia J. Burrows
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：July 22, 2015
• 年：2015
• 卷：137
• 期：28
• 页码：9053-9060
• 全文大小：603K
• ISSN：1520-5126

文摘

Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the 伪-hemolysin (伪-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (i鈥?i>t) traces when the i-motif interacts with 伪-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of 伪-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5鈥?25-mer poly-2鈥?deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the 伪-HL nanopore, a feature that is not easily achievable by other methods.