Emergent kinetics of in vitro transcription from interactions of T7 RNA polymerase and DNA

The in vitro transcription reaction (IVT) is of growing importance for the manufacture of RNA vaccines and therapeutics. While the kinetics of the microscopic steps of this reaction (promoter binding, initiation, and elongation) are well studied, the rate law of overall RNA synthesis that emerges from this system is unclear. In this work, we show that a model that incorporates both initiation and elongation steps is essential for describing trends in IVT kinetics in conditions relevant to RNA manufacturing. In contrast to previous reports, we find that the IVT reaction can be either initiation- or elongation-limited depending on solution conditions. This initiation-elongation model is also essential for describing the effect of salts, which disrupt polymerase-promoter binding, on transcription rates. Polymerase-polymerase interactions during elongation are incorporated into our modeling framework and found to have nonzero but unidentifiable effects on macroscopic transcription rates. Finally, we develop an extension of our modeling approach to quantitatively describe and experimentally evaluate RNA- and DNA-templated mechanisms for the formation of double-stranded RNA (dsRNA) impurities. We show experimental results that indicate that an RNA-templated mechanism is not appropriate for describing macroscopic dsRNA formation in the context of RNA manufacturing.