Brunet Erika, Alberti Patrizia, Perrouault Loïc, Babu Ravindra, Wengel Jesper, Giovannangeli Carine
Laboratoire de Biophysique, Museum National d'Histoire Naturelle USM 503, CNRS UMR 5153, INSERM U 565, Paris, France.
J Biol Chem. 2005 May 20;280(20):20076-85. doi: 10.1074/jbc.M500021200. Epub 2005 Mar 10.
Triplex-forming oligonucleotides (TFOs), as DNA-binding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine.oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect.
三链形成寡核苷酸（TFOs）作为识别特定序列的DNA结合分子，为理解DNA上发生的过程及其相关功能提供了独特的潜力。它们也是用于定位作用于DNA的普遍存在分子（如抗癌药物）的强大DNA识别元件。包括TFOs在内的DNA编码读取分子进一步发展的一个先决条件是它们在细胞环境中形成复合物的能力：它们的结合亲和力必须与DNA相关蛋白的结合亲和力相当。为了实现这一目标，必须开发化学修饰的TFOs。在这项工作中，我们展示了含鸟嘌呤的锁核酸修饰的TFOs（TFO/LNAs）的三链形成特性（动力学和热力学）及细胞活性。在模拟生理条件下，与未修饰的TFO或针对相同寡嘧啶-寡嘌呤序列的嘧啶TFO/LNA相比，这些TFO/LNAs极大地增强了三链稳定性，主要是通过降低解离速率常数并带来熵增。我们通过基于三链的机制在体外和细胞环境中，在亲本磷酸二酯寡核苷酸未表现出任何抑制作用的条件下，证明了它们的生物活性。
