High-salt transcription of DNA cotethered with T7 RNA polymerase to beads generates increased yields of highly pure RNA

Abstract

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High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs

“…As noted above and previously demonstrated ( 26 ), one would expect the rebinding of product RNA that initiates self-primed extension to be salt sensitive (and independent of the promoter that drives its extension). To confirm this prediction here, we repeated the above experiment, in Figure 3 , with a DNA sequence (panel A) encoding an RNA previously shown to generate self-primed extension products ( 10 ).…”

Quality by Design for enabling RNA platform production processes

“…could further decrease this risk by redefining the potential IVT design space and notably increase the salt concentration [66]. Recent innovations in the field also recommend performing IVT and enzymatic capping reactions at higher temperatures either to prevent dsRNA formation or to decrease capping enzyme concentration [64,67].…”

“…The expansion of these models to RNA impurities, and particularly dsRNA, would further refine process design and control strategy. Our increased mechanistic understanding of the different dsRNA formation mechanisms is particularly promising [65,66,101]. The use of mechanistic modeling can be advantageous in an initial data-scarce environment to direct experimental effort as well as ease technology transfer or adapt the process to product innovations [102].…”

“…Multiple chromatographic modeling options can also be rapidly explored. For example, studies on CaptoCore ligands and shells detail their binding, adsorption, and diffusion properties and enable the development of accurate predictive models characterizing the capture of proteins as a function of their size [66,92,105].…”

Double-stranded RNA reduction by chaotropic agents during in vitro transcription of messenger RNA

“…As a result, a sharp yield loss was observed since magnesium is an essential cofactor for RNA polymerases. 14 , 15 Three recent reports recruited competing oligonucleotides, 16 co-tethered DNA promoter and RNA polymerase, 17 and high temperature 15 to reduce dsRNA formation during IVT, respectively. However, custom reagents or elevated temperature impose significant economic and technical burdens for scale-up mRNA manufacturing.…”