Roche Kulmbach GmbH, D-95326 Kulmbach, Germany.
J Chromatogr A. 2011 Aug 19;1218(33):5609-17. doi: 10.1016/j.chroma.2011.06.057. Epub 2011 Jun 22.
Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.
小干扰 RNA（siRNA）作为一种新的治疗模式，正在特异性抑制靶基因表达方面崭露头角。siRNA 通常由两条互补的单链寡核糖核苷酸退火形成。与通过变性色谱方法测定未杂交单链的纯度相比，杂交双链的特性分析具有一定挑战性。本文报道了一种非变性的离子对反相（IP-RP）色谱方法，该方法使用紫外和质谱检测，能够分离最佳双链（仅全长单链）和非最佳双链变体（包含短链、长链和 2',5'-异构体）。研究了不同退火条件对 siRNA 组成的影响。优化的退火条件导致最佳双链显著增加，而双链总量保持不变。与其他 IP-RP 缓冲体系相比，本文报道的非变性方法显示出更高的质谱灵敏度和更好的分离效率。该方法可用于治疗性双链核酸（如 siRNA）的过程控制和放行测试。
