Preparative

Pellicular anion-exchange chromatography applied to RNAi assays for monitoring strand stoichiometry and RNA stability.

Mar 24 2011

Author: J. R. Thayer, Srinivasa Rao, Christopher Pohl (Dionex Corporation) Yansheng Wu, and Mark Angelino (Archemix Corporation). on behalf of Unassigned Independent Article

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Abstract
RNAi products require both single strand and duplex impurity assays. Duplex assays should allow titration of guide or passenger strands that may be in excess. These assays are chromatographic, and typically employ two steps: First, as is  common to other single-stranded therapeutics, denaturing conditions must resolve both guide and passenger strand, and thus may reveal impurities formed during the annealing process. Next, native conditions, where the duplex is stable, are used to resolve the duplex and both single strands from one another.
This allows analysis of possible duplex impurities, such as those arising from annealed impurities from the guide, passenger, or both strands. RNAi component strands, or those of other therapeutic modes, may harbor one or two phosphorothioate linkages. These introduce diastereoisomers that may impact the RNAi stability or efficacy. Resolution of the diastereoisomers allows assessment of the influence of each in the process, and where the separation can be accomplished at mg levels, can support bioanalytical identification of the contribution of each diastereomer. We describe here simple anion-exchange chromatographic approaches to accomplish these separations.

Introduction:
Among the continually increasing numbers of RNA therapeutic modes, four have been identified as clinically promising. These are Antisense oligonucleotides (ONs) [1], Ribozymes [2], Aptamers [3], and RNA interference (RNAi ) [4,5] ONs. While several new non-coding RNA forms have been described recently, these four therapeutic modes show promise. The RNAi approach conscripts a subset of microRNA (miRNA) processing machinery, allowing exogenously supplied RNA of specific sequence to stimulate turnover of accordingly-specific mRNA [6]. Where the activity (or defect) in a specific protein is known to produce or support a disease state, turnover of the mRNA encoding that specific protein will suppress progression of that disease, or it symptoms.

Numerous examples of gene-product “Knock- Down” have been described, ranging from control of Cholesterol levels [7] to suppression of Ebola Virus replication [8]. Understandably, this has stimulated intense interest in RNAi as a new therapeutic model.
 

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