Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Raiding nature’s toolbox to discover novel RNA polymerases from diverse phage (#432)

Jenni A Hayward 1 , Manisha Treeby 1 , Gaetan Burgio 2 , Gavin J Knott 1
  1. Biochemistry And Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VICTORIA, Australia
  2. John Curtin School of Medical Research, Australian National University, Acton, ACT, Australia

The COVID-19 pandemic highlighted the importance of RNA production for mRNA-based vaccines on the global stage. The current workhorse used to synthesize RNA in vitro is a single subunit DNA-dependent RNA polymerase (RNAP) enzyme from T7 bacteriophage. However, T7 RNAP has many qualities that are less than ideal for the large-scale production of mRNA. Specifically, T7 RNAP has a propensity to generate immunostimulatory dsRNA, prematurely terminate transcription of long and/or structured RNA and make non-templated additions to the 3’ end of the RNA. These attributes therefore limit the portfolio of RNA that can be reliably produced by T7 RNAP and necessitate extensive purification to remove unwanted harmful by-products. Here, we describe our approach to harness the evolved diversity of novel phage RNAPs to overcome these limitations and expand the repertoire of enzymes available for mRNA synthesis.