Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides
Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical metho...
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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 |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/15476286.2024.2363029 |
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| author | Francis Combes Thanh-Huong Bui Frida J. Pettersson Sjoerd Hak |
| author_facet | Francis Combes Thanh-Huong Bui Frida J. Pettersson Sjoerd Hak |
| author_sort | Francis Combes |
| collection | DOAJ |
| description | Production and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3’-5’ exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques. |
| format | Article |
| id | doaj-art-35a9f18a99204cc880a0c631d0e17b3a |
| institution | Kabale University |
| issn | 1547-6286 1555-8584 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj-art-35a9f18a99204cc880a0c631d0e17b3a2024-12-11T07:34:00ZengTaylor & Francis GroupRNA Biology1547-62861555-85842024-12-0121166567210.1080/15476286.2024.2363029Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotidesFrancis Combes0Thanh-Huong Bui1Frida J. Pettersson2Sjoerd Hak3Department 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, NorwayProduction and storage of synthetic mRNA can introduce a variety of byproducts which reduce the overall integrity and functionality of mRNA vaccines and therapeutics. mRNA integrity is therefore designated as a critical quality attribute which must be evaluated with state-of-the-art analytical methods before clinical use. The current study first demonstrates the effect of heat degradation on transcript translatability and then describes a novel enzymatic approach to assess the integrity of conventional mRNA and long self-amplifying mRNA. By first hybridizing oligo-T to the poly(A) tail of intact mRNA and subsequently digesting the unhybridized RNA fragments with a 3’-5’ exoribonuclease, individual nucleotides can be selectively released from RNA fragments. The adenosine-based fraction of these nucleotides can then be converted into ATP and detected by luminescence as a sensitive indicator of mRNA byproducts. We developed a polynucleotide phosphorylase (PNPase)-based assay that offers fast and sensitive evaluation of mRNA integrity, regardless of its length, thus presenting a novel and fully scalable alternative to chromatographic-, electrophoresis-, or sequencing-based techniques.https://www.tandfonline.com/doi/10.1080/15476286.2024.2363029mRNAbyproductsPNPaseassayhigh-throughput |
| spellingShingle | Francis Combes Thanh-Huong Bui Frida J. Pettersson Sjoerd Hak Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides RNA Biology mRNA byproducts PNPase assay high-throughput |
| title | Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| title_full | Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| title_fullStr | Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| title_full_unstemmed | Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| title_short | Rapid and scalable detection of synthetic mRNA byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| title_sort | rapid and scalable detection of synthetic mrna byproducts using polynucleotide phosphorylase and polythymidine oligonucleotides |
| topic | mRNA byproducts PNPase assay high-throughput |
| url | https://www.tandfonline.com/doi/10.1080/15476286.2024.2363029 |
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