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
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DOI：
10.1093/nar/gki726
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Giovannangeli, C
中科院分区：
文献类型：
--
作者：
Brunet, E;Corgnali, M;Giovannangeli, C
三链体形成寡核苷酸（TFO）是序列特异性干扰DNA相关生物学功能的有力工具。含（A/T，G）的TFO比含（T，C）的TFO更常用于细胞中，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后者活性较低。在这项工作中，我们研究了增强含（T，C）的TFO的DNA结合的可能性，旨在达到细胞活性;为此，我们使用了锁核酸修饰的TFO（TFO/LNA）与嵌入剂吖啶衍生物的5 '-缀合。在体外，用TFO-吖啶缀合物形成稳定的三链体：通过在37 ° C和中性pH下的SPIR测量，发现解离平衡常数在纳摩尔范围内，并且三链体半衰期类似于10小时（与未缀合的TFO/ LNA相比长50倍）。此外，为了进一步了解含（T，C）TFO/LNA的DNA结合，在不同pH值下进行杂交研究：与pH降低相关的三链体稳定化主要是由于较慢的解离过程。最后，在细胞环境中评价嘧啶TFO/LNA的生物活性：对于吖啶缀合的TFO/LNA，其在类似于0.1 μ M的浓度下发生（或对于未缀合的TFO/LNA，其在类似于2 μ M的浓度下发生），而相应的磷酸二酯TFO是无活性的，并且其被证明是三链体介导的。
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 SPIR measurements at 37 degrees C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life similar to 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 similar to 0.1 mu M for acridine-conjugated TFO/LNA (or similar to 2 mu M for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated.