Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography

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• 作者：Bernhard Noll¹ ; ^a ; ^{Bernhard.noll@roche.com"" rel=""nofollow} ; ^{bernhard_noll@gmx.de"" rel=""nofollow} ; Stephan Seiffert¹ ; ^a ; Hans-Peter Vornlocher^a ; Ingo Roehl^a
• 关键词：Sirna ; Oligonucleotide ; Non-denaturing ; Duplex analysis ; HPLC ; Mass spectrometry
• 刊名：Journal of Chromatography A
• 出版年：2011
• 出版时间：19 August 2011
• 年：2011
• 卷：1218
• 期：33
• 页码：5609-5617
• 全文大小：442 K

文摘

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.