Published online 27 July 2005
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Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities
Laboratoire de Biophysique, Museum national d'Histoire naturelle USM 0503, CNRS UMR 5153, INSERM U 565 43 rue Cuvier, 75005 Paris, France 1Dipartimento di Scienze e Tecnologie Biomediche, Universita degli Studi di Udine 33100 Udine, Italy 2Centre de Biophysique Moléculaire, CNRS UPR4301 Rue Charles Sadron, 45071 Orléans Cedex 2, France 3Nucleic Acid Center, Department of Chemistry, University of Southern Denmark Campusvej 55, DK-5230 Odense M, Denmark
*To whom correspondence should be addressed. Tel: +33 1 40 79 37 11; Fax: +33 1 40 79 37 05; Email: giovanna{at}cimrs1.mnhn.fr
Received May 13, 2005. Revised July 5, 2005. Accepted July 5, 2005.
Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5'-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life 
10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations 0.1 µM for acridine-conjugated TFO/LNA (or 2 µM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.
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