Brunet Erika, Corgnali Maddalena, Perrouault Loïc, Roig Victoria, Asseline Ulysse, Sørensen Mads D, Babu B Ravindra, Wengel Jesper, Giovannangeli Carine
Laboratoire de Biophysique, Museum national d'Histoire naturelle, USM 0503, CNRS UMR 5153, INSERM U 565 43 rue Cuvier, 75005 Paris, France.
Nucleic Acids Res. 2005 Jul 27;33(13):4223-34. doi: 10.1093/nar/gki726. Print 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 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.
三链形成寡核苷酸（TFOs）是用于序列特异性干扰与DNA相关的生物学功能的强大工具。含（A/T，G）的TFOs在细胞中比含（T，C）的TFOs更常用，尤其是富含C的序列；实际上，非共价嘧啶三链体的低细胞内稳定性使得后者活性较低。在这项工作中，我们研究了增强含（T，C）的TFOs与DNA结合的可能性，旨在实现细胞活性；为此，我们使用了锁定核酸修饰的TFOs（TFO/LNAs）并结合一种嵌入剂（一种吖啶衍生物）的5'-共轭。在体外，TFO-吖啶共轭物形成了稳定的三链体：通过在37℃和中性pH下的表面等离子体共振（SPR）测量，解离平衡常数在纳摩尔范围内，三链体半衰期约为10小时（与未共轭的TFO/LNA相比长50倍）。此外，为了进一步了解含（T，C）的TFO/LNAs与DNA的结合，在不同pH值下进行了杂交研究：与pH降低相关的三链体稳定性增强主要是由于解离过程较慢。最后，在细胞环境中评估了嘧啶TFO/LNAs的生物学活性：吖啶共轭的TFO/LNA在浓度约为0.1 microM时发生作用（未共轭的TFO/LNA约为2 microM），而相应的磷酸二酯TFO则无活性，并且证明其是三链体介导的。
