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Bacterial RNA Polymerase

Bacterial RNA Polymerase

Unlike eukaryotes, bacteria use a single RNA Polymerase (RNAP) to transcribe all genes. The different subunits of bacterial RNAPhave distinct functions. The multisubunit structure of the bacterial RNAP helps the enzyme to maintain catalytic function, facilitate assembly, interact with DNA and RNA, and self-regulate its activity.
In most genes, the transcription site is a single base present upstream of the coding sequence. Though RNAP is a catalytically efficient enzyme, it does not recognize...

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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

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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.

International Journal of Biological Macromolecules

|April 3, 2026

Summary

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.

Keywords:

Directed evolution T7 RNA polymerase mRNA

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Published on: February 26, 2019

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.