Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities
Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities
Published online 27 July 2005
Erika Brunet, Maddalena Corgnali1, Lo?c Perrouault, Victoria Roig2, Ulysse Asseline2, Mads D. S?rensen3, B. Ravindra Babu3, Jesper Wengel3 and Carine Giovannangeli
Nucleic Acids Research 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 ?M for acridine-conjugated TFO/LNA (or 2 ?M for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.
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