NOXXON Pharma AG, Max-Dohrn-Strasse 8-10, D-10589 Berlin, Germany.
Nucleic Acids Res. 2011 Nov;39(21):e147. doi: 10.1093/nar/gkr776. Epub 2011 Sep 24.
The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5'-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control.
验证基于核酸的治疗药物序列是药物开发过程中的一个重要步骤。随着核酸分子的长度和耐核酸酶性的增加，与序列鉴定相关的挑战也会增加，后者是治疗性寡核苷酸的一个重要属性。我们描述了 Spiegelmers 的序列确定方法， Spiegelmers 是天然存在的 RNA 的对映异构体，具有很高的抗酶降解能力。 Spiegelmer 测序是通过将标记或半抗原附着在寡核苷酸的 5'端，并以受控的方式化学降解分子，生成片段，然后使用液相色谱-质谱法对这些片段进行分离和鉴定来实现的。然后从这些片段中推导出 Spiegelmer 的序列。本文展示了两种不同 Spiegelmers（NOX-E36 和 NOX-A12）的实例，并使用 NOX-E36 错配对照显示了该方法的特异性。
