Related Experiment Video
Updated: Apr 5, 2026
DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Published on: December 29, 2021
Minimizing dsRNA impurity generation through structure-function-guided directed evolution of T7 RNA polymerase
Wei He1, Jingchao Xi2, Xiaoqin Wu3
1College of Forestry, Nanjing Forestry University, Nanjing, 210037, China; Vazyme Biotech Co., Ltd., Nanjing, 210000, China.
Abstract:
The presence of dsRNA, a prominent pathogen-associated molecular pattern, poses a significant challenge for mRNA therapeutics due to its potent activation of innate immune responses. Extensive research has been conducted in the field in recent years, leading to the discovery of various strategies for dsRNA reduction. Among these approaches, T7 RNA polymerase (T7 RNAP) engineering stands out as the most efficacious method. However, comprehensive research elucidating the detailed mechanisms underlying the generation of dsRNA during in vitro transcription catalyzed by T7 RNAP remains lacking. The results of the present study reveal the crucial involvement of the RNA exit tunnel in both transcription termination and dsRNA byproduct formation. Based on this relationship, we engineered T7 RNAP using phage-assisted non-continuous evolution. The resulting engineered polymerases contain at least one mutation at the RNA exit tunnel, thereby providing additional validation. Moreover, we introduce a T7 RNAP harboring double mutations, M183E + I210V, minimizing the dsRNA content to an unprecedented level, with >99% of dsRNA eliminated, making it an ideal candidate for the development of next-generation mRNA therapeutics.
Insights
Engineering T7 RNA polymerase (T7 RNAP) reduces double-stranded RNA (dsRNA) byproducts in mRNA therapeutics. Mutations in the RNA exit tunnel minimize dsRNA, enhancing mRNA therapeutic development.
Area of Science:
- Molecular Biology
- Biotechnology
- RNA Therapeutics
Background:
- Double-stranded RNA (dsRNA) is a pathogen-associated molecular pattern that activates innate immunity, posing challenges for mRNA therapeutics.
- Current strategies for dsRNA reduction in mRNA synthesis often involve T7 RNA polymerase (T7 RNAP) engineering.
Purpose of the Study:
- To elucidate the mechanisms of dsRNA generation during T7 RNAP-catalyzed in vitro transcription.
- To engineer T7 RNAP variants with significantly reduced dsRNA byproduct formation for improved mRNA therapeutics.
Main Methods:
- Investigated the role of the RNA exit tunnel in transcription termination and dsRNA formation.
- Employed phage-assisted non-continuous evolution to engineer T7 RNAP variants.
- Introduced specific mutations within the RNA exit tunnel of T7 RNAP.
Main Results:
- The RNA exit tunnel is critical for both transcription termination and dsRNA byproduct generation.
- Engineered T7 RNAP variants with mutations in the RNA exit tunnel showed reduced dsRNA.
- A double mutant T7 RNAP (M183E + I210V) eliminated over 99% of dsRNA.
Conclusions:
- The RNA exit tunnel of T7 RNAP is a key determinant of dsRNA byproduct formation.
- Engineered T7 RNAP variants, particularly the M183E + I210V double mutant, significantly minimize dsRNA content.
- These engineered polymerases are highly promising for developing next-generation mRNA therapeutics with enhanced safety and efficacy.
More Related Videos
08:55RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points
Published on: May 29, 2020
11:37Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
