Monia B P, Johnston J F, Ecker D J, Zounes M A, Lima W F, Freier S M
Department of Molecular and Cellular Biology, ISIS Pharmaceuticals, Carlsbad, California 92008.
J Biol Chem. 1992 Oct 5;267(28):19954-62.
A biological reporter gene assay was employed to determine the crucial parameters for maximizing selective targeting of a Ha-ras codon 12 point mutation (G----T) using phosphorothioate antisense oligonucleotides. We have tested a series of oligonucleotides ranging in length between 5 and 25 bases, each centered around the codon 12 point mutation. Our results indicate that selective targeting of this point mutation can be achieved with phosphorothioate antisense oligonucleotides, but this selectivity is critically dependent upon oligonucleotide length and concentration. The maximum selectivity observed in antisense experiments, 5-fold for a 17-base oligonucleotide, was closely predicted by a simple thermodynamic model that relates the fraction of mutant to wild type target bound as a function of oligonucleotide concentration and affinity. These results suggest thermodynamic analysis of oligonucleotide/target interactions is useful in predicting the specificity that can be achieved by an antisense oligonucleotide targeted to a single base point mutation.
采用生物报告基因分析法来确定使用硫代磷酸酯反义寡核苷酸使Ha-ras密码子12点突变（G----T）的选择性靶向最大化的关键参数。我们测试了一系列长度在5至25个碱基之间的寡核苷酸，每个都围绕密码子12点突变。我们的结果表明，硫代磷酸酯反义寡核苷酸可以实现对该点突变的选择性靶向，但这种选择性关键取决于寡核苷酸的长度和浓度。在反义实验中观察到的最大选择性，即17碱基寡核苷酸为5倍，通过一个简单的热力学模型得到了密切预测，该模型将突变体与野生型靶标结合的比例作为寡核苷酸浓度和亲和力的函数。这些结果表明，寡核苷酸/靶标相互作用的热力学分析有助于预测针对单个碱基点突变的反义寡核苷酸所能实现的特异性。
