反义寡核苷酸药物癌泰得的定性分析方法研究

Study on the Method of the Qualitative Analysis of Phosphorothioate Oligonucleotide ‘Cantide’

癌泰得 (ACTCACTCAGGCCTCAGACT)为端粒酶表达抑制活性反义寡核苷酸 .为了探讨其定性检测手段 ,通过阴离子交换高效液相色谱和毛细管凝胶电泳分析方法 ,确定了该硫代寡核苷酸以及与其有关的短序列和部分未被硫代类似物的保留时间 ,并分析了不同混合物样品 .结果表明 ,阴离子交换高效液相色谱对硫代寡核苷酸骨架上的差异非常敏感 ,可很好地分离长度相同的硫代和未完全硫代类似物 ,并且随未被硫代磷酸基数目增加 ,保留时间依次缩短 .阴离子交换高效液相色谱对硫代寡核苷酸的长度不敏感 ,不能分离相差一个碱基的硫代寡核苷酸 .毛细管凝胶电泳可很好地分离长度相差一个碱基的硫代寡核苷酸 ,不能分离同长硫代和部分硫代寡核苷酸 .高效液相色谱结合毛细管凝胶电泳可有效地确定癌泰得的纯度和修饰程度 .

Cantide (ACTCACTCAGGCCTCAGACT) is an antisense inhibitor against the activity of telomerase.To study the method of the qualitative analysis,anion exchange high performance liquid chromatography and capillary gel electrophoresis were used.The phos\|phorothioate oligonucleotide and its analogs of deletion sequences as well as partial phosphodiester compounds were analyzed.The results showed that anion exchange HPLC was sensitive to phosphorothioate modification and full phosphorothioate and partial phosphodiester oligonucleotides of the same length could be resolved.The retention time is decreased with the increasing number of phosphodiester bonds.Anion exchange HPLC was relatively insensitive to the length of phosphorothioate oligonucleotides,n and n\|1 sequence could not be resolved.CGE was excellent for separating the deletion sequences from the full\|length sequence,but it could not separate phosphorothioate and partial phosphodiester oligonucleotides of the same length.Purity and modified degree of phosphorothioate oligonucleotides could be determined by anion exchange HPLC and CGE.