Optimization of repeated-batch transcription for RNA production.
A major obstacle to large-scale RNA production is the high raw material cost. This work focuses on reducing the cost of RNA produced by in vitro transcription. RNA can be produced by transcription from DNA templates immobilized on solid supports such as agarose beads, with yields comparable to traditional solution-phase transcription. The advantage of immobilized DNA is that the templates can be recovered from the reaction and reused in multiple rounds, eliminating unnecessary disposal. Additionally, approximately 50% of the original RNA polymerase added to the reaction is also recovered in active form with the DNA and can be used for further rounds of repeated-batch transcription. Thus, adding only a fraction of the first-round enzyme concentration to subsequent rounds is sufficient for maintaining yields comparable to batch reactions for many rounds, with lowered cost. Results for two different DNA templates support a simplified model for repeated-batch transcription, based on the previous work of Davis and Breckenridge (J Biotechnol 1999;71:25-37). The model successfully predicts the yields for several of rounds of repeated-batch transcription using various enzyme addition schemes, and it was used to optimize the process by reducing the cost of raw materials per amount of RNA produced by 40-70%.
Breckenridge
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NC
Department of Chemical Engineering, University of Colorado, Boulder, Colorado 80309-0424, USA.
Davis
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RH
D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology / Q000379:methods*; D008969:Molecular Sequence Data; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012313:RNA