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Updated: Jun 24, 2026
09:26
DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation
Published on: December 29, 2021
Functional comparison of SP6 RNA polymerase and T7 RNA polymerase
Julia Gutbrod1, Svenja Hehn1, Antonia Bangnowski1
1Department of Chemistry, University of Konstanz, Konstanz Research School Chemical Biology, Konstanz, Germany.
Plos One
|June 22, 2026
Summary
This study compares T7 and SP6 RNA polymerases for therapeutic RNA synthesis. SP6 RNA polymerase shows promise for incorporating modified nucleotides and produces less double-stranded RNA, improving RNA quality.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Development
Background:
- RNA-based therapeutics, including COVID-19 vaccines, utilize in vitro transcription (IVT) with bacteriophage RNA polymerases (RNAP).
- Challenges in IVT include incorporating modified nucleotides and reducing double-stranded RNA (dsRNA) by-products.
- Engineering efforts for T7 RNAP are extensive, but SP6 RNAP has been less studied, lacking direct functional comparisons.
Purpose of the Study:
- To systematically compare wild-type (wt) T7 and SP6 RNAP.
- To evaluate their efficiency in incorporating modified nucleotides (2'-F-UTP and 2'-O-methyl-UTP).
- To assess dsRNA formation during IVT for both polymerases.
Main Methods:
- Comparative analysis of wt T7 and SP6 RNAP.
- Evaluation of engineered RNAP variants.
- In vitro transcription (IVT) using modified nucleotides 2'-F-UTP and 2'-O-methyl-UTP.
- Quantification of dsRNA by-products.
Main Results:
- T7 RNAP generally yielded more RNA for long transcripts, with a T7 variant excelling in 2'-F-UTP incorporation.
- SP6 RNAP was uniquely capable of synthesizing a ~760 nt transcript using 2'-OMe-UTP.
- Wild-type SP6 RNAP produced significantly less dsRNA compared to wild-type T7 RNAP during IVT.
Conclusions:
- T7 and SP6 RNAP possess distinct and complementary strengths for RNA synthesis.
- SP6 RNAP variants show potential for synthesizing mRNA with bulky modifications.
- Reduced dsRNA formation by SP6 RNAP offers an advantage for improving RNA quality in therapeutics.
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