# Directed Evolution of T7 RNA Polymerase Minimizes dsRNA By-product and Enables High-Fidelity mRNA Synthesis for Demanding Therapeutic Applications

## TL;DR

Researchers engineered a new version of T7 RNA polymerase that reduces harmful by-products and improves mRNA quality for medical use.

## Contribution

A novel T7 RNA polymerase variant, M30, with reduced dsRNA by-products and higher catalytic efficiency for therapeutic mRNA synthesis.

## Key findings

- M30 shows a 10-fold increase in catalytic efficiency compared to wild-type T7 RNAP at 37°C.
- M30 produces approximately 10-fold less dsRNA by-products, reducing immunogenicity in mRNA therapies.
- M30-synthesized mRNAs enable efficient protein expression in human cells and mice.

## Abstract

T7 RNA polymerase (T7 RNAP) is the most widely used enzyme for synthesizing therapeutic mRNA. However, RNA transcribed by T7 RNAP often contains double-stranded RNA (dsRNA) by-products that trigger innate immune responses and complicate purification. Here, we report an engineered T7 RNAP variant, M30, which exhibits higher catalytic efficiency and reduced dsRNA by-product formation. M30 was developed through 4 rounds of directed evolution using an ultrahigh-throughput aptamer-based fluorescence-activated droplet sorting system. M30 displays a 10-fold increase in catalytic efficiency over wild-type T7 RNAP at 37 °C, along with markedly enhanced thermostability and approximately 10-fold lower production of dsRNA by-products. mRNAs synthesized with M30 achieve efficient protein expression in human cells and in mice, while eliciting reduced immunogenicity compared with mRNAs produced by wild-type T7 RNAP. Biophysical assays and structural analyses suggest that these improvements result from increased DNA template binding affinity and decreased RNA binding affinity. Together, these features make M30 a promising catalyst for high-quality therapeutic mRNA production.