Engineering of T7 DNA-dependent RNA polymerase with activity at elevated temperature

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Bacteriophage T7 RNA polymerase (T7 RNAP) is crucial for in vitro transcription (IVT) of synthetic mRNA.
• Undesired double-stranded RNA (dsRNA) byproducts in IVT trigger immune responses and reduce mRNA quality.
• Wild-type T7 RNAP has limited stability at elevated temperatures, hindering efforts to reduce dsRNA by increasing reaction temperature.

Purpose of the Study:

• To engineer T7 RNAP variants with enhanced thermotolerance for improved IVT.
• To minimize dsRNA byproduct formation during mRNA synthesis.
• To enable robust transcription at elevated temperatures.

Main Methods:

• Utilized the Protein Repair One Stop Shop (PROSS) web server for protein engineering.
• Employed four crystal structures of T7 RNAP (promoter, initiation, elongation complexes) as input for PROSS.
• Combined mutations from multi-structure PROSS designs to create PROSS Combined Designs (PCDs).

Main Results:

• PCD9 demonstrated full-length transcription activity up to 48 °C, unlike wild-type T7 RNAP.
• At 48 °C, PCD9 produced kilobase-length transcripts with no detectable dsRNA.
• Wild-type T7 RNAP showed significant activity loss at 48 °C and produced dsRNA at 37 °C.

Conclusions:

• Multi-structure PROSS design successfully yielded a thermotolerant T7 RNAP variant (PCD9).
• The engineered T7 RNAP exhibits improved performance at elevated temperatures and reduced dsRNA byproduct formation.
• Designing for stability across multiple structural states is crucial for engineering dynamic enzymes like T7 RNAP.