In Vitro Transcription

Slater, Robert

doi:10.1007/978-1-4615-9799-5_12

Cited by 4 publications

(1 citation statement)

References 36 publications

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“…Despite the T7 polymerase being the most essential component in order to initiate IVT 24 , other factors influence optimal IVT productivity. Magnesium ions are known to influence the catalytic activity of T7 polymerase 25 .…”

Section: Discussionmentioning

confidence: 99%

Design-of-Experiments In Vitro Transcription Yield Optimization of Self-Amplifying RNA

Samnuan

Blakney

McKay

et al. 2021

Preprint

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Self-amplifying RNA (saRNA) vaccines are able to induce a higher antigen-specific immune response with a more cost-effective and rapid production process compared to plasmid DNA vaccines. saRNAs are synthesized through in vitro transcription (IVT) however; this process has mainly been optimized for relatively short mRNAs. Here, we optimized the IVT process for long saRNAs, approximately 9.4 kb through a design of experiment (DoE) approach to produce a maximal RNA yield and validated the optimal IVT method on various sizes of RNA. We found that magnesium has the highest impact on RNA yield with acetate ions enabling a higher yield than chloride ions. In addition, the interaction between magnesium and nucleoside triphosphates (NTPs) is highly essential for IVT. Further addition of sodium acetate (NaOAc) during IVT provided no added benefit in RNA yield. Moreover, pyrophosphatase was not essential for productive IVT. The optimal IVT method can be used to synthesize different lengths of RNA. These findings emphasize the ability to synthesize high quality and quantity of saRNA through IVT and that the optimal amount of each component is essential for their interactions to produce a high RNA yield.

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Section: Discussionmentioning

confidence: 99%