通过毛细管凝胶电泳对反义寡核苷酸进行稳定性测量。
Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.
作者信息
Bruin G J, Börnsen K O, Hüsken D, Gassmann E, Widmer H M, Paulus A
机构信息
Corporate Analytical Research, Ciba, Basel, Switzerland.
出版信息
J Chromatogr A. 1995 Aug 11;709(1):181-95. doi: 10.1016/0021-9673(95)00231-b.
The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented.
将反义寡核苷酸用作潜在药物的方法是基于短化学修饰寡核苷酸与互补的细胞DNA或RNA序列杂交。一个关键问题是化学修饰的反义寡核苷酸在细胞环境中的稳定性。在一个模型系统中，通过毛细管凝胶电泳（CGE）评估了对各种核酸酶的抗性。对于一些样品，基质辅助激光解吸电离质谱（MALDI-MS）被用作额外的分析工具来进行稳定性测量。使用CGE，可以在不同孵育时间后跟踪寡聚物中单核苷酸的酶促降解。10% T聚丙烯酰胺凝胶可为长度在5至30个碱基之间的寡核苷酸提供基线分辨率。讨论了特定核酸酶浓度和反义寡核苷酸结构对切割反应的动力学影响。此外，还描述了一种简单的脱盐方法，以提高该方法的进样效率和灵敏度。给出了化学修饰的19聚体反义寡核苷酸的测量示例。
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