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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

A padlock oligonucleotide for labelling plasmids

We have recently developped a new method for labelling supercoiled plasmid DNA using a triple helical complex intermediate. The central part of a triple-helix forming oligonucleotide (TFO) binds specifically to an oligopurine-oligopyrimidine sequence located on the plasmid. The ends of this TFO then hybridize to a template oligonucleotide and are joined through the action of T4 DNA ligase, thus forming a padlock oligonucleotide attached to the supercoiled plasmid (1).

Formation of this structure has been carried out with different types of triple helices. When a GT oligonucleotide that form a triple helix only in the presence of a triple helix stabilizing agent (TSA) (2) was used, a restriction enzyme inhibition assay showed that the interaction between the catenated TFO and the plasmid could be modulated by the TSA (3). In vitro transcription experiments with T3 RNA polymerase confirmed these results as the padlock oligonucleotide was able to stop transcription only in the presence of the TSA.

Padlocks could also be formed with modified oligonucleotides. We showed that a biotinylated oligonucleotide which had been catenated to a supercoiled plasmid could bind streptavidin. A streptavidin-ferritin complex was used for sequence specific labelling of supercoiled DNA in electron microscopy experiments.

References and Footnotes
  1. Escude, C., Garestier, T. and Helene, C. (1999) Padlock oligonucleotides for duplex DNA based on sequence-specific triple helix formation Proc. Natl. Acad. Sci. U S A, 96(19), 10603-7.
  2. Escude, C., Sun, J. S., Nguyen, C. H., Bisagni, E., Garestier, T. and Helene, C. (1996) Ligand-induced formation of triple helices with antiparallel third strands containing G and T Biochemistry, 35(18), 5735-40.
  3. Roulon, T., Helene, C. and Escude, C., (2001) A ligand-modulated padlock-oligonucleotide for supercoiled plasmids Angew. Chem. Int. Ed. Engl., 40(8), in press.

T. Roulon (1), E. Le Cam (2), T. Garestier (1), C. Helene (1) and C. Escude (1).

(1) Laboratoire de Biophysique, Museum National d'Histoire Naturelle, 43 rue Cuvier 75231 PARIS CEDEX 05, France.
(2) Laboratoire de Microscopie Cellulaire et Moleculaire, Institut Gustave Roussy PR2, 39, rue Camille Desmoulins 94805 VILLEJUIF CEDEX, France.
Email: roulon@cimrs1.mnhn.fr