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Nucleic Acids Research, 2002, Vol. 30, No. 3 e12
© 2002 Oxford University Press

Padlock oligonucleotides as a tool for labeling superhelical DNA

Thibaut Roulon, Dominique Coulaud1, Etienne Delain1, Eric Le Cam1, Claude Hélène and Christophe Escudé*

Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, INSERM U201, CNRS UMR8646, 43 Rue Cuvier, F-75231 Paris Cedex 05, France and 1Laboratoire de Microscopie Cellulaire et Moléculaire, CNRS UMR 8532, Institut Gustave Roussy, Rue Camille Desmoulins, F-94805 Villejuif Cedex, France

Labeling of a covalently closed circular double-stranded DNA was achieved using a so-called ‘padlock oligonucleotide’. The oligonucleotide was targeted to a sequence which is present in the replication origin of phage f1 and thus in numerous commonly used plasmids. After winding around the double-stranded target DNA sequence by ligand-induced triple helix formation, a biotinylated oligonucleotide was circularized using T4 DNA ligase and in this way became catenated to the plasmid. A gel shift assay was developed to measure the extent of plasmid modification by the padlock oligonucleotide. A similar assay showed that a modified supercoiled plasmid was capable of binding one streptavidin molecule thanks to the biotinylated oligonucleotide and that this binding was quantitative. The catenated complex was visualized by electron and atomic force microscopies using streptavidin conjugates or single strand-binding proteins as protein tags for the padlock oligonucleotide. This method provides a versatile tool for plasmid functionalization which offers new perspectives in the physical study of supercoiled DNA and in the development of improved vectors for gene therapy.

* To whom correspondence should be addressed. Tel: +33 1 40 79 37 74; Fax: +33 1 40 79 37 05; Email: escude{at}mnhn.fr


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