ArticleNucleic acids research2024
DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants.
Article in Nucleic acids research, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Cold-adapted RNA polymerase fromProceedings of the National Academy of Sciences of the United States of America · 2026Article
- The current landscape of mRNA therapy and the strategies for mRNA purification and dsRNA removal.Journal of biomedical science · 2026Review
- Modifying the upstream open reading frames of cellulase gene enhances cellulase production inSynthetic and systems biotechnology · 2026Article
- Macromolecular crowding alters transcription: real-time measurements with SYBR Green II.Research square · 2026Article
- Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement.Nucleic acids research · 2026Article
- Directed Evolution of T7 RNA Polymerase Minimizes dsRNA By-product and Enables High-Fidelity mRNA Synthesis for Demanding Therapeutic Applications.Research (Washington, D.C.) · 2026Article
- Article
- Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced.Molecules (Basel, Switzerland) · 2024Article
Corrections and comments
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Authors and funding
5 authors.
Funding
Abstract
The remarkable success of messenger RNA (mRNA)-based vaccines has underscored their potential as a novel biotechnology platform for vaccine development and therapeutic protein delivery. However, the single-subunit RNA polymerase from bacteriophage T7 widely used for in vitro transcription is well known to generate double-stranded RNA (dsRNA) by-products that strongly stimulate the mammalian innate immune response. The dsRNA was reported to be originated from self-templated RNA extension or promoter-independent transcription. Here, we identified that the primary source of the full-length dsRNA during in vitro transcription is the DNA-terminus-initiated transcription by T7 RNA polymerase. Guanosines or cytosines at the end of DNA templates enhance the DNA-terminus-initiated transcription. Moreover, we found that aromatic residues located at position 47 in the C-helix lead to a significant reduction in the production of full-length dsRNA. As a result, the mRNA synthesized using the T7 RNA polymerase G47W mutant exhibits higher expression efficiency and lower immunogenicity compared to the mRNA produced using the wild-type T7 RNA polymerase.
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Registered trials
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