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Separation of small interfering RNA stereoisomers using reversed-phase ion-pairing chromatography
Journal article   Peer reviewed

Separation of small interfering RNA stereoisomers using reversed-phase ion-pairing chromatography

Li Li, Tony Leone, Joe P. Foley and Christopher J. Welch
Journal of Chromatography A, v 1500, pp 84-88
02 Jun 2017
DOI: https://doi.org/10.1016/j.chroma.2017.04.008
PMID: 28449879
Featured in Collection :   UN Sustainable Development Goals @ Drexel

Abstract

Desulfurization Diastereomer Ion-pairing reversed-phase liquid chromatography Phosphorothioate Small interfering RNA Stereoisomer
•Baseline separation of stereoisomers of a siRNA duplex was reported using UHPLC.•On-column dissociation of the duplex using TEAA/ACN is critical to the separation.•The method can resolve oxidation products of the sulfur-modified siRNA duplex. Small interfering RNA (siRNA) therapeutics have received significant interest in recent years owing to their ability to elicit sequence-specific gene knockdown and subsequent suppression of protein expression. Chemical modifications can improve the hydrolytic stability and susceptibility of siRNAs to enzymatic degradation. One commonly used modification to improve hydrolytic stability, the replacement of the native phosphodiester linkage with a phosphorothioate moiety, introduces an additional phosphorous stereocenter into the molecule, resulting in the formation of diastereomers. The chromatographic separation of stereoisomeric siRNAs containing such phosphothioates is a challenging problem, especially when multiple phosphothioates are present within a modified siRNA duplex giving rise to multiple stereoisomers. In this study, we report an investigation into the use of an ion-pairing reversed phase UHPLC (or IP-RP UHPLC) method for the baseline separation of closely related diastereomers of an Apo-B gene targeting siRNA duplex under denaturing conditions. The related siRNA species consist of two diastereomers from the sense strand and the two pairs of diastereomers from the antisense strand. Key chromatographic parameters critical to stereoisomer separation are highlighted, including the structure of the ion-pairing agent, chemical composition of the stationary phase, and organic modifier. The method was applied to the separation of an siRNA stressed with iodine and demonstrated satisfactory selectivity for the parent siRNA and the desulfurization product.

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Collaboration types
Industry collaboration
Domestic collaboration
Web of Science research areas
Biochemical Research Methods
Chemistry, Analytical
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