A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide
van der Oost, J
Nucleic Acids Research
Oxford University Press (OUP)
The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interactions.
Netherlands Organisation for Scientific Research and the Netherlands Institute for Space Research [ALW-GO-PL/ 08-08]; NWO Vidi grant [864.11.005 to S.J.J.B.]. Funding for open access charge: Microbiology department/ Wageningen UR library.
This is the final version of the article. Available from the publisher via the DOI in this record.
Vol. 41, e111
PubMed Central ID
Place of publication