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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| Format: | Article |
| Language: | English |
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Elsevier
2025-06-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253125001039 |
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| author | Nathaniel E. Clark Mateusz Kozarski Sinem Demirel Asci Jurgen Van den Heuvel Matt R. Schraut Roger A. Winters Kelley Kearns Thomas C. Scanlon Senne Dillen |
| author_facet | Nathaniel E. Clark Mateusz Kozarski Sinem Demirel Asci Jurgen Van den Heuvel Matt R. Schraut Roger A. Winters Kelley Kearns Thomas C. Scanlon Senne Dillen |
| author_sort | Nathaniel E. Clark |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-6eb3bbe4e43b455f858d40e11da44ebd |
| institution | DOAJ |
| issn | 2162-2531 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-6eb3bbe4e43b455f858d40e11da44ebd2025-08-20T02:58:34ZengElsevierMolecular Therapy: Nucleic Acids2162-25312025-06-0136210254910.1016/j.omtn.2025.102549Removal of dsRNA byproducts using affinity chromatographyNathaniel E. Clark0Mateusz Kozarski1Sinem Demirel Asci2Jurgen Van den Heuvel3Matt R. Schraut4Roger A. Winters5Kelley Kearns6Thomas C. Scanlon7Senne Dillen8Repligen, 16 Cavendish CT, Lebanon, NH 03766, USA; Corresponding author: Nathaniel E Clark, Repligen, 37 Cavendish CT, Lebanon, NH 03766, USA.etherna, Galileilaan 19, 2845 Niel, Belgiumetherna, Galileilaan 19, 2845 Niel, Belgiumetherna, Galileilaan 19, 2845 Niel, BelgiumRepligen, 16 Cavendish CT, Lebanon, NH 03766, USARepligen, 16 Cavendish CT, Lebanon, NH 03766, USARepligen, 16 Cavendish CT, Lebanon, NH 03766, USARepligen, 16 Cavendish CT, Lebanon, NH 03766, USAetherna, Galileilaan 19, 2845 Niel, Belgium; Corresponding author: Senne Dillen, etherna, Galileilaan 19, 2845 Niel, Belgium.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.http://www.sciencedirect.com/science/article/pii/S2162253125001039MT: Oligonucleotides: Therapies and Applicationsdouble-stranded RNAmessenger RNAcircular RNAself-amplifying RNAin vitro transcription |
| spellingShingle | Nathaniel E. Clark Mateusz Kozarski Sinem Demirel Asci Jurgen Van den Heuvel Matt R. Schraut Roger A. Winters Kelley Kearns Thomas C. Scanlon Senne Dillen Removal of dsRNA byproducts using affinity chromatography Molecular Therapy: Nucleic Acids MT: Oligonucleotides: Therapies and Applications double-stranded RNA messenger RNA circular RNA self-amplifying RNA in vitro transcription |
| title | Removal of dsRNA byproducts using affinity chromatography |
| title_full | Removal of dsRNA byproducts using affinity chromatography |
| title_fullStr | Removal of dsRNA byproducts using affinity chromatography |
| title_full_unstemmed | Removal of dsRNA byproducts using affinity chromatography |
| title_short | Removal of dsRNA byproducts using affinity chromatography |
| title_sort | removal of dsrna byproducts using affinity chromatography |
| topic | MT: Oligonucleotides: Therapies and Applications double-stranded RNA messenger RNA circular RNA self-amplifying RNA in vitro transcription |
| url | http://www.sciencedirect.com/science/article/pii/S2162253125001039 |
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