title: Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography.
abstract: 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.
authors: Roche Kulmbach GmbH, D-95326 Kulmbach, Germany. Bernhard.noll@roche.com
authors: Bernhard
authors: B
authors: Noll
authors: Stephan
authors: S
authors: Seiffert
authors: Hans-Peter
authors: HP
authors: Vornlocher
authors: Ingo
authors: I
authors: Roehl
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D034741:RNA, Small Interfering