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An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

ABSTRACT: In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex purification processes, using methods such as reversed-phase high-performance liquid chromatography, to yield safe and effective mRNA-based medicines. To minimize the need for downstream purification processes, we rationally and computationally engineered a double mutant of T7 RNAP that produces substantially less immunostimulatory RNA during IVT compared with wild-type T7 RNAP. The resulting mutant allows for a simplified production process with similar mRNA potency, lower immunostimulatory content and quicker manufacturing time compared with wild-type T7 RNAP. Herein, we describe the computational design and development of this improved T7 RNAP variant.

SUBMITTER: Dousis A

PROVIDER: S-EPMC10110463 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

Dousis Athanasios A Ravichandran Kanchana K Hobert Elissa M EM Moore Melissa J MJ Rabideau Amy E AE

Nature biotechnology 20221110 4

In vitro transcription (IVT) is a DNA-templated process for synthesizing long RNA transcripts, including messenger RNA (mRNA). For many research and commercial applications, IVT of mRNA is typically performed using bacteriophage T7 RNA polymerase (T7 RNAP) owing to its ability to produce full-length RNA transcripts with high fidelity; however, T7 RNAP can also produce immunostimulatory byproducts such as double-stranded RNA that can affect protein expression. Such byproducts require complex puri ...[more]

PMID: 36357718

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Project description:RNA polymerase (RNAP) is the primary machine responsible for transcription. Its ability to distinguish between correct (cognate) and incorrect (noncognate) nucleoside triphosphates (NTPs) is important for fidelity control in transcription. In this work, we investigated the substrate selection mechanism of T7 RNAP from the perspective of energetics. The dissociation free energies were determined for matched and unmatched base pairs in the preinsertion complex using the umbrella sampling method. A clear hydrogen-bond-rupture peak is observed in the potential of mean force curve for a matched base pair, whereas no such peaks are present in the position of mean force profiles for unmatched ones. The free-energy barrier could prevent correct substrates from being separated from the active site. Therefore, when NTPs diffuse into the active site, correct ones will stay for chemistry once they establish effective base pairing contacts with the template nucleotide, whereas incorrect ones will be withdrawn from the active site and rejected back to solution. This result provides an important energy evidence for the substrate selection mechanism of RNAP. Then we elucidated energetics and molecular details for correct NTP binding to the active site of the insertion complex. Our observations reveal that strong interactions act on the triphosphate of NTP to constrain its movement, whereas relatively weak interactions serve to position the base in the correct conformation. Triple interactions, hydrophobic contacts from residues M635 and Y639, base stacking from the 3' RNA terminal nucleotide, and base pairing from the template nucleotide act together to position the NTP base in a catalytically competent conformation. At last, we observed that incorrect NTPs cannot be as well-stabilized as the correct one in the active site when they are misincorporated in the insertion site. It is expected that our work can be helpful for comprehensively understanding details of this basic step in genetic transcription.

Dataset Information

An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

Publications

An engineered T7 RNA polymerase that produces mRNA free of immunostimulatory byproducts.

Similar Datasets

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