Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Tokyo 162-8601, Japan.
J Biochem. 2012 Jul;152(1):17-26. doi: 10.1093/jb/mvs049. Epub 2012 May 3.
Since pyrimidine motif triplex DNA is unstable at physiological neutral pH, triplex stabilization at physiological neutral pH is important for improvement of its potential to be applied to various methods in vivo, such as repression of gene expression, mapping of genomic DNA and gene-targeted mutagenesis. For this purpose, we studied the thermodynamic and kinetic effects of a chemical modification, 2'-O,4'-C-ethylene bridged nucleic acid (ENA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological neutral pH. Thermodynamic investigations indicated that the modification achieved more than 10-fold increase in the binding constant of the triplex formation. The increased number of the modification in TFO enhanced the increased magnitude of the binding constant. On the basis of the obtained thermodynamic parameters, we suggested that the remarkably increased binding constant by the modification may result from the increased stiffness of TFO in the unbound state. Kinetic studies showed that the considerably decreased dissociation rate constant resulted in the observed increased binding constant by the modification. We conclude that ENA modification of TFO could be a useful chemical modification to promote the triplex formation under physiological neutral condition, and may advance various triplex formation-based methods in vivo.
由于嘧啶基三联体 DNA 在生理中性 pH 下不稳定，因此在生理中性 pH 下稳定三联体对于提高其在体内各种方法中的应用潜力非常重要，例如基因表达的抑制、基因组 DNA 的作图和基因靶向诱变。为此，我们研究了化学修饰（2'-O，4'-C-亚乙基桥接核酸（ENA）修饰三链形成寡核苷酸（TFO））对嘧啶基三联体形成的热力学和动力学影响在生理中性 pH 下。热力学研究表明，修饰使三联体形成的结合常数增加了 10 倍以上。TFO 中修饰的数量增加增强了结合常数的增加幅度。基于获得的热力学参数，我们认为修饰引起的结合常数的显着增加可能是由于未结合状态下 TFO 的刚性增加所致。动力学研究表明，解离速率常数的显著降低导致了修饰引起的观察到的结合常数的增加。我们得出结论，ENA 修饰 TFO 可能是一种有用的化学修饰方法，可促进生理中性条件下的三联体形成，并可能推进体内各种基于三联体形成的方法。
