Engineering of T7 DNA-dependent RNA polymerase with activity at elevated temperature
Svenja Hehn, Julia Gutbrod, Maxi Gutjahr, Andreas Marx
Abstract
Bacteriophage T7 RNA polymerase (T7 RNAP) is a key enzyme for in vitro transcription (IVT) and plays a central role in the production of synthetic mRNA for research and therapeutic applications. However, IVT frequently generates double-stranded RNA (dsRNA) as an undesired by-product, which can trigger innate immune responses and compromise mRNA quality. Increasing reaction temperatures reduces dsRNA formation, however, the wild-type T7 RNA polymerase exhibits limited stability under such conditions.
Introduction
T7 RNA polymerase (T7 RNAP), a single subunit DNA dependent RNA polymerase from bacteriophage T7, is a fundamental tool in molecular biology because of its high efficiency and robust activity in transcribing DNA into RNA [1]. Its applications span synthetic biology, controlled gene expression, and the production of RNA for therapeutic purposes, including mRNA based therapeutics [2–4].
Materials and method
General information
T7 RNAP variants were ordered as gene fragments from Azenta. BL21 competent cells were used for expression and XL10 gold cells were used for cloning procedures. Templates for in vitro transcription experiments and primers were ordered from Biomers. NTPs were purchased from Carl Roth. RNase inhibitors were obtained from Jena Bioscience.
Results and discussion
Multi-structure PROSS design identifies a thermotolerant T7 RNAP variant
The PROSS webserver was applied separately to four crystal structures of T7 RNAP that represent promoter (PDB 1CEZ), initiation (PDB 1QLN), and elongation complexes (PDB 1MSW and 1H38) [25–29]. For each structure, nine PROSS designs were obtained. Comparison of the designs across the four structures for each design index revealed that only a subset of substituted positions was shared.
Conclusion
Multi-structure PROSS design yielded PCD9, a T7 RNAP variant with increased thermotolerance that extends the useful transcription temperature window to 48 °C under optimized buffer conditions and reduces dsRNA byproducts when transcribing a kilobase-length scale template. PCD9 is more thermotolerant than wt T7 RNAP but less thermotolerant than M0 + G788A under the same assay and reaction conditions, providing a useful option for in vitro transcription workflows operating at moderately elevated temperatures.
Acknowledgments
We gratefully acknowledge support by the Konstanz Research School Chemical Biology. We thank Rooa Moslemany for assistance with experimental work.
Citation: Hehn S, Gutbrod J, Gutjahr M, Marx A (2026) Engineering of T7 DNA-dependent RNA polymerase with activity at elevated temperature. PLoS One 21(7): e0353775. https://doi.org/10.1371/journal.pone.0353775
Editor: Jian Xu, Kyushu University Faculty of Agriculture Graduate School of Bioresource and Bioenvironmental Sciences: Kyushu Daigaku Nogakubu Daigakuin Seibutsu Shigen Kankyo Kagakufu, JAPAN
Received: March 2, 2026; Accepted: June 28, 2026; Published: July 20, 2026
Copyright: © 2026 Hehn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: European Commission (project: NEWmRNA; grant no. 965135).
Competing interests: The authors declare no competing interests.
