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Nucleic Acids Research Advance Access published online on September 28, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm529
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© 2007 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.

Methods Online

Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide

Masahito Hayashi1,* and Yoshie Harada1,2

1The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613 and 2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan

*To whom correspondence should be addressed. Tel: +81 3 4463 7588; Fax: +81 3 3823 1247; Email: mhayashi{at}rinshoken.or.jp

Received March 5, 2007. Revised June 5, 2007. Accepted June 25, 2007.

Ethidium bromide (EtBr) is the conventional intercalator for visualizing DNA. Previous studies suggested that EtBr lengthens and unwinds double-stranded DNA (dsDNA). However, no one has observed the unwinding of a single dsDNA molecule during intercalation. We developed a simple method to observe the twisting motions of a single dsDNA molecule under an optical microscope. A short dsDNA was attached to a glass surface of a flow chamber at one end and to a doublet bead as a rotation marker at the other end. After the addition and removal of EtBr, the bead revolved in opposite directions that corresponded to the unwinding and rewinding of a dsDNA, respectively. The amount of intercalating EtBr was estimated from the revolutions of the bead. EtBr occupied 57% of base pairs on a single dsDNA at 1 mM of EtBr, indicating that EtBr molecules could bind at contiguous sites to each other. The isotherm of intercalation showed that negative cooperativity existed between adjoining EtBr molecules. The association constant of EtBr and dsDNA (1.9 (±0.1) x 105 M–1) was consistent with that of previous results. Our system is useful to investigate the twisting of a single dsDNA interacting with various chemicals and biomolecules.


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