title: Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities.
abstract: 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 degrees C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life approximately 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 approximately 0.1 microM for acridine-conjugated TFO/LNA (or approximately 2 microM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.
authors.affiliation: Laboratoire de Biophysique, Museum national d'Histoire naturelle, USM 0503, CNRS UMR 5153, INSERM U 565 43 rue Cuvier, 75005 Paris, France.
authors.forename: Erika
authors.initials: E
authors.lastname: Brunet
authors.forename: Maddalena
authors.initials: M
authors.lastname: Corgnali
authors.forename: Loïc
authors.initials: L
authors.lastname: Perrouault
authors.forename: Victoria
authors.initials: V
authors.lastname: Roig
authors.forename: Ulysse
authors.initials: U
authors.lastname: Asseline
authors.forename: Mads D
authors.initials: MD
authors.lastname: Sørensen
authors.forename: B Ravindra
authors.initials: BR
authors.lastname: Babu
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Carine
authors.initials: C
authors.lastname: Giovannangeli
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007364:Intercalating Agents / Q000737:chemistry* / Q000494:pharmacology*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011743:Pyrimidines / Q000737:chemistry*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D004268:DNA-Binding Proteins; D007364:Intercalating Agents; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011743:Pyrimidines; C477371:locked nucleic acid; C051802:triplex DNA; D004247:DNA