Functional comparison of SP6 RNA polymerase and T7 RNA polymerase

Abstract

RNA-based therapeutics have emerged as a powerful class of drugs, highlighted by the rapid development and success of COVID-19 vaccines. Therapeutic RNA synthesis relies on in vitro transcription (IVT), most commonly using bacteriophage RNA polymerases (RNAP) such as T7 RNAP and SP6 RNAP. However, efficient incorporation of modified nucleotides and the reduction of double-stranded RNA (dsRNA) by-products to improve pharmacokinetic properties and reduce immunostimulatory effects remain major challenges. 

Introduction

In vitro transcribed RNA has become a central component of modern RNA therapeutics, with applications ranging from mRNA vaccines [1,2] and cancer vaccines [3,4] to protein replacement [5,6] and gene regulation [7,8]. Research has spiked, especially on the development of mRNA vaccines, which have proven to be a powerful tool against viral pathogens like SARS CoV 2 [1,2].

Materials and method

Generation of SP6 RNAP mutants F and FA

Point mutations at positions Y631 and H779 were successively introduced into the SP6 RNAP wt polymerase gene carried on the pET21b vector using site-directed mutagenesis. E. coli XL10-Gold cells were transformed to identify successfully mutated variants. Colonies were screened by Sanger sequencing and plasmids containing the desired mutation were used to transform BL21 DE3 for protein expression. 

Results and discussion

Structural comparison of T7 and SP6 RNAP and generation of SP6 RNAP mutants
The lack of structural data available for SP6 RNAP limits direct structure-guided engineering of this enzyme. To address this limitation, the amino acid sequence of SP6 RNAP was submitted to AlphaFold2 for structure prediction. Among the five generated models, the structure with the highest predicted confidence was selected to perform structural alignment with the T7 RNAP intermediate complex (PDB: 3E2E) [36].

Conclusion

In this study, we performed a systematic comparison of T7 and SP6 RNAP and their engineered variants with respect to nucleotide analogue incorporation and dsRNA by-product formation. While T7 RNAP remains the most widely used enzyme for IVT, our results demonstrate that SP6 RNAP represents a viable and, in some respects, advantageous alternative for the synthesis of modified RNA. 

Acknowledgments

We gratefully acknowledge support by the Konstanz Research School Chemical Biology.
Citation: Gutbrod J, Hehn S, Bangnowski A, Fritz E, Marx A (2026) Functional comparison of SP6 RNA polymerase and T7 RNA polymerase. PLoS One 21(6): e0351567. https://doi.org/10.1371/journal.pone.0351567

Editor: Hyun Ho Park, Chung-Ang University, KOREA, REPUBLIC OF

Received: March 2, 2026; Accepted: May 28, 2026; Published: June 22, 2026

Copyright: © 2026 Gutbrod 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 manuscript and its Supporting Information files.

Funding: EU Horizon 2020 programme (NEWmRNA, Grant No. 965135). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.