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Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation
Alesia A Levanova1, Mirka Lampi1, Kiira Kalke2
1Molecular and Integrative Biosciences Research Programme, Biological and Environmental Sciences, University of Helsinki, Viikinkaari 9, FI-00014 Helsinki, Finland.
Pharmaceuticals (Basel, Switzerland)
|February 26, 2022
Summary
Asymmetrical flow field-flow fractionation (AF4) gently purifies antiviral small interfering RNA (siRNA) molecules. This method offers higher recovery and retains biological activity, proving effective against viral infections.
Area of Science:
- RNA biology and medicine
- Macromolecular separation science
Background:
- RNA molecules show potential for treating viral infections.
- Advanced purification methods are needed for RNA processing under non-denaturing conditions.
- Asymmetrical flow field-flow fractionation (AF4) gently separates macromolecules based on diffusion coefficients.
Purpose of the Study:
- Develop an AF4 method for purifying enzymatically produced antiviral small interfering RNA (siRNA).
- Evaluate AF4's potential for separating short single-stranded (ss) and double-stranded (ds) RNA molecules.
- Assess the purity, recovery, and biological activity of AF4-purified siRNA.
Main Methods:
- Utilized asymmetrical flow field-flow fractionation (AF4) for RNA molecule separation.
- Compared AF4 with anion-exchange chromatography for siRNA purification.
- Assessed siRNA toxicity and biological activity in mammalian cell cultures.
Main Results:
- AF4 successfully separated monomeric ssRNA from dsRNA and multimeric ssRNA.
- Developed AF4 method purified 27-nt siRNAs from toxic dsRNA substrate with ~70% recovery.
- AF4-purified siRNAs were non-toxic and inhibited herpes simplex virus 1 replication.
Conclusions:
- AF4 is a viable method for gentle and efficient purification of short RNA molecules.
- AF4 offers superior recovery compared to traditional methods like anion-exchange chromatography.
- AF4-purified siRNAs maintain biological activity and are suitable for therapeutic applications.
