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%.

Modeling of repeated-batch transcription for production of RNA.

Enzymatic transcription for in vitro production of ribonucleic acids (RNA) is typically carried out in small batch reactors which suffer from low yields and high costs due to the formation of abortive RNA transcripts and the disposal of the DNA template and polymerase enzyme after a single use. This work considers repeated-batch transcription in which the DNA template molecules are immobilized on beads which are recovered and reused in multiple rounds. Some of the enzyme binds to the DNA template and is also recovered and reused. A model of this process is presented which employs equilibrium binding between the enzyme and template and which includes a first-order sequential deactivation of the enzyme. The model predicts the yields of RNA product and aborts for each round of repeated-batch transcription with no DNA addition after the first round and only partial enzyme replacement. The yield of RNA product per substrate (nucleoside triphosphates) generally decreases in subsequent rounds, whereas the yields of RNA product per enzyme and per template increase due to their reuse. Experimental data are presented which confirm the model and which show how the model parameters are obtained. A cost analysis shows that the cost of RNA production can be reduced by more than 50% for the system tested by employing an optimum number of rounds of repeated-batch transcription.