A sequence-to-function model to predict T7 transcription rates and redesign T7 expression systems with lowered production of immunogenic RNA byproducts

<jats:p> T7 RNA polymerase is widely used to produce RNA using a canonical T7 promoter; however, it will also bind to low-affinity sites to generate cryptic transcription and produce RNA byproducts, which reduce full-length mRNA purity and yield. When manufacturing therapeutic RNAs for clinical applications, RNA byproducts must be removed using costly downstream purification and can cause adverse immunogenicity. To predict T7 transcription rates and reduce cryptic transcription, we designed 11588 T7 promoters and measured their mRNA levels, spanning a 6300-fold range within in vitro transcription reactions. We developed the T7 Promoter Calculator, a sequence-to-function machine learning model that predicts the T7 transcription rate on arbitrary DNA sequence across a 500-fold range with high accuracy (R <jats:sup>2</jats:sup> = 0.80), accounting for both core and flanking motif sequences. We combined the model with generative design to remove low-affinity T7 sites from a therapeutic T7 expression system, resulting in a 2-fold increase in full-length mRNA purity. The automated design of T7 expression systems to remove undesired RNA byproducts increases mRNA purity and lowers downstream separation costs, while reducing adverse immunogenicity. </jats:p>