Removal of dsRNA byproducts using affinity chromatography
Double-stranded RNA (dsRNA) molecules are immunogenic byproducts of in vitro transcription of single-stranded RNA (ssRNA). Removal of dsRNA from ssRNA is difficult because the byproducts have similar sizes, sequences, and charges to the desired ssRNA. Here, we describe a dsRNA-specific affinity resi...
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| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125001039 |
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| Summary: | Double-stranded RNA (dsRNA) molecules are immunogenic byproducts of in vitro transcription of single-stranded RNA (ssRNA). Removal of dsRNA from ssRNA is difficult because the byproducts have similar sizes, sequences, and charges to the desired ssRNA. Here, we describe a dsRNA-specific affinity resin that selectively removes dsRNA from ssRNA. Affinity purification reduced dsRNA levels by >100-fold, to as low as ∼0.00007% w/w of total mRNA, with no negative impact on RNA integrity. The purified RNA, synthesized with standard nucleotides, induced no inflammatory response in a reporter cell line assay designed to measure innate immune responses. Purified RNA induced greater protein expression and healthier cells. The immunogenicity of the affinity-purified RNA with standard nucleotides compares favorably to RNA synthesized with modified nucleotides and purified with cellulose or reverse-phase high-performance liquid chromatography (HPLC). dsRNA affinity purification provides a facile and scalable solution to the problem of immunogenic dsRNA byproducts in transcribed RNA. This approach will improve quality and safety of RNA vaccines and therapeutics. |
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| ISSN: | 2162-2531 |