Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription
RNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP)...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2022-12-01
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| Series: | RNA Biology |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/15476286.2022.2139113 |
| _version_ | 1797403983272214528 |
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| author | Heng Xia Bingbing Yu Yixin Jiang Rui Cheng Xueling Lu Hui Wu Bin Zhu |
| author_facet | Heng Xia Bingbing Yu Yixin Jiang Rui Cheng Xueling Lu Hui Wu Bin Zhu |
| author_sort | Heng Xia |
| collection | DOAJ |
| description | RNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4–25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3’-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use. |
| first_indexed | 2024-03-09T02:46:27Z |
| format | Article |
| id | doaj.art-a0b4c153499749698a56c482a2043fde |
| institution | Directory Open Access Journal |
| issn | 1547-6286 1555-8584 |
| language | English |
| last_indexed | 2024-03-09T02:46:27Z |
| publishDate | 2022-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | RNA Biology |
| spelling | doaj.art-a0b4c153499749698a56c482a2043fde2023-12-05T16:09:51ZengTaylor & Francis GroupRNA Biology1547-62861555-85842022-12-011911130114210.1080/15476286.2022.21391132139113Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcriptionHeng Xia0Bingbing Yu1Yixin Jiang2Rui Cheng3Xueling Lu4Hui Wu5Bin Zhu6Huazhong University of Science and TechnologyHuazhong University of Science and TechnologyHuazhong University of Science and TechnologyHuazhong University of Science and TechnologyHuazhong University of Science and TechnologyHuazhong University of Science and TechnologyHuazhong University of Science and TechnologyRNA research and applications are underpinned by in vitro transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out in vitro RNA synthesis at low temperatures (4–25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3’-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for in vivo use.http://dx.doi.org/10.1080/15476286.2022.2139113dsrnasgrnamrna medicinet7 rna polymerasein vitro transcription |
| spellingShingle | Heng Xia Bingbing Yu Yixin Jiang Rui Cheng Xueling Lu Hui Wu Bin Zhu Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription RNA Biology dsrna sgrna mrna medicine t7 rna polymerase in vitro transcription |
| title | Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription |
| title_full | Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription |
| title_fullStr | Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription |
| title_full_unstemmed | Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription |
| title_short | Psychrophilic phage VSW-3 RNA polymerase reduces both terminal and full-length dsRNA byproducts in in vitro transcription |
| title_sort | psychrophilic phage vsw 3 rna polymerase reduces both terminal and full length dsrna byproducts in in vitro transcription |
| topic | dsrna sgrna mrna medicine t7 rna polymerase in vitro transcription |
| url | http://dx.doi.org/10.1080/15476286.2022.2139113 |
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