An engineered T7 RNA polymerase for efficient co-transcriptional capping with reduced dsRNA byproducts in mRNA synthesis
Mathew Miller, Oscar Alvizo, Scott Baskerville, Avinash Chintala, Chinping Chng, Justin P. Dassie, Jonathan Dorigatti, Gjalt W. Huisman, Stephan Jenne, S. S Kadam, Neil Leatherbury, Stefan Lutz, Melissa Mayo, Arpan Mukherjee, Antoinette Sero, Stuart Sundseth, Jonathan Penfield, James N. Riggins, Xiyun Zhang
Abstract
transcription (IVT) using wild-type T7 RNA polymerase generates undesirable double-stranded RNA (dsRNA) byproducts that elicit adverse host immune responses and are difficult to remove at large scale. To overcome these challenges, we have engineered a novel RNA polymerase, T7-68, that co-transcriptionally incorporates both di- and tri-nucleotide cap analogs with high efficiency, even at reduced cap analog concentrations. We also demonstrate that IVT products generated with T7-68 have reduced dsRNA content.