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Nucleic Acids Research Advance Access published online on December 26, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn968
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Structural Biology

Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity

Ji Wook Shim, Qiulin Tan and Li-Qun Gu*

Department of Biological Engineering, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA

*To whom correspondence should be addressed. Tel: +1 573 882 2057; Fax: +1 573 884 4232; Email: gul{at}missouri.edu

Received August 29, 2008. Revised November 12, 2008. Accepted November 16, 2008.

Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K+ > NH4+ ~ Ba2+ > Cs+ ~ Na+ > Li+, and G-quadruplex was not detected in Mg2+ and Ca2+. Ba2+ can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation–DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K+-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na+- and Li+-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na+-quadruplex folds and unfolds most rapidly, while the Li+-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein–aptamer interactions.


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