Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

• Published in: Nucleic acids research Vol. 33; no. 13; pp. 4223 - 4234
• Main Authors: Brunet, Erika, Corgnali, Maddalena, Perrouault, Loïc, Roig, Victoria, Asseline, Ulysse, Sørensen, Mads D., Babu, B. Ravindra, Wengel, Jesper, Giovannangeli, Carine
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.01.2005; Oxford Publishing Limited (England)
• Subjects: DNA - chemistry; DNA - metabolism; DNA-Binding Proteins - antagonists & inhibitors; HeLa Cells; Humans; Hydrogen-Ion Concentration; Intercalating Agents - chemistry; Intercalating Agents - pharmacology; Oligonucleotides; Oligonucleotides, Antisense - chemistry; Oligonucleotides, Antisense - metabolism; Oligonucleotides, Antisense - pharmacology; Pyrimidines - chemistry; Transcription, Genetic - drug effects
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gki726

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.