International journal of biological macromoleculesApr 3, 2026

Reducing unwanted double-stranded RNA by improving T7 RNA polymerase based on its structure and function

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Engineered T7 RNA polymerase variants, especially M183E + I210V, sharply reduced dsRNA byproducts during in vitro transcription.

Evidence

This structure-function-guided directed-evolution and in vitro transcription study engineered T7 RNAP variants and tested dsRNA byproduct formation.

Caveat

The abstract reports an enzyme-platform experiment, not therapeutic mRNA manufacturing performance or immune outcomes in animals or humans.

Simplified

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.

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Reducing unwanted double-stranded RNA by improving T7 RNA polymerase based on its structure and function

Abstract

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