DNA-terminus-dependent transcription by T7 RNA polymerase and its C-helix mutants
Yu, B.; Chen, Y.; Yan, Y.; Zhu, B.
Show abstract
The remarkable success of 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) byproducts 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 interfere with the binding of T7 RNA polymerase to DNA termini, leading 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.
Matching journals
The top 4 journals account for 50% of the predicted probability mass.
Similar papers in this journal
- A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase 98%
- Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli 94%
- Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq 94%
Similar papers in this journal
- Impact of an Oxidative RNA Lesion on in vitro Replication Catalyzed by SARS-CoV-2 RNA-dependent RNA Polymerase 96%
- The 3' end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by RNA-dependent RNA polymerase 96%
- Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq 95%
Similar papers in this journal
- An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production 94%
- Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen 93%
- Development of a mini-replicon-based reverse-genetics system for rice stripe tenuivirus 93%
Similar papers in this journal
- Synthesis of low immunogenicity RNA with high-temperature in vitro transcription. 96%
- Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity 94%
- Chemi-Northern: a versatile chemiluminescent northern blot method for analysis and quantitation of RNA molecules. 93%
"Similar papers" are the closest papers from that journal in the model's embedding space. They show what the match is built on, but the ranking comes mostly from a classifier over the whole training set, not from these examples alone.