Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts
ABSTRACTThe current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | RNA Biology |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2024.2321764 |
| _version_ | 1797293802125262848 |
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| author | Combes Francis Pettersson Frida J Bui Thanh-Huong Molska Alicja Komissarov Artem Parot Jérémie Borgos Sven Even |
| author_facet | Combes Francis Pettersson Frida J Bui Thanh-Huong Molska Alicja Komissarov Artem Parot Jérémie Borgos Sven Even |
| author_sort | Combes Francis |
| collection | DOAJ |
| description | ABSTRACTThe current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages. |
| first_indexed | 2024-03-07T21:21:06Z |
| format | Article |
| id | doaj.art-9dfddd53d58d4585b4d1802f65af5b56 |
| institution | Directory Open Access Journal |
| issn | 1547-6286 1555-8584 |
| language | English |
| last_indexed | 2024-03-07T21:21:06Z |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj.art-9dfddd53d58d4585b4d1802f65af5b562024-02-27T12:18:56ZengTaylor & Francis GroupRNA Biology1547-62861555-85842024-12-012111610.1080/15476286.2024.2321764Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproductsCombes Francis0Pettersson Frida J1Bui Thanh-Huong2Molska Alicja3Komissarov Artem4Parot Jérémie5Borgos Sven Even6Department of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayDepartment of Biotechnology and Nanomedicine, SINTEF, Trondheim, NorwayABSTRACTThe current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.https://www.tandfonline.com/doi/10.1080/15476286.2024.2321764In vitro transcriptionureaVSW3RNA polymerasetranscript impuritiesmRNA byproducts |
| spellingShingle | Combes Francis Pettersson Frida J Bui Thanh-Huong Molska Alicja Komissarov Artem Parot Jérémie Borgos Sven Even Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts RNA Biology In vitro transcription urea VSW3 RNA polymerase transcript impurities mRNA byproducts |
| title | Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts |
| title_full | Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts |
| title_fullStr | Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts |
| title_full_unstemmed | Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts |
| title_short | Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts |
| title_sort | urea supplementation improves mrna in vitro transcription by decreasing both shorter and longer rna byproducts |
| topic | In vitro transcription urea VSW3 RNA polymerase transcript impurities mRNA byproducts |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2024.2321764 |
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