Hollenstein Marcel, Hipolito Christopher J, Lam Curtis H, Perrin David M
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver BC, V6T 1Z1, Canada.
Nucleic Acids Res. 2009 Apr;37(5):1638-49. doi: 10.1093/nar/gkn1070. Epub 2009 Jan 19.
The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M(2+))-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 +/- 0.026) min(-1). A pH rate profile analysis revealed pK(a)'s of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M(2+)-free conditions at 37 degrees C.
修饰脱氧核酶的筛选是拓展催化核酸化学和催化性质的一项重要工作。此类例子很少，迄今为止，尚无将三种不同修饰碱基同时引入以实现催化活性的例子。在此，分别带有阳离子胺、咪唑和阳离子胍的dCTP、dATP和dUTP在DNA模板上进行酶促聚合，以筛选一种高度功能化的脱氧核酶，称为脱氧核酶9 - 86，它在生理条件下能催化单个核糖（胞嘧啶）磷酸二酯键发生（不依赖M(2+)的）自我切割，速率常数为(0.134 ± 0.026) min(-1)。pH速率曲线分析显示其pK(a)值分别为7.4和8.1，这与一般酸碱催化相符。胍阳离子的存在使得切割能在比仅含胺和咪唑的脱氧核酶所观察到的温度显著更高的温度下发生。定性地说，脱氧核酶9 - 86展现了前所未有的合成功能组合，而定量地说，在无M(2+)条件下于37℃进行研究时，它表现出了任何自我切割核酸所报道的最高值之一。
