Structure-switching DNA templates for the rational and predictable control of cell-free transcription

We report here a strategy to control cell-free in vitro transcription of RNA by the rational design of synthetic structure-switching DNA templates. These templates are programmed to undergo a conformational change between an open (OFF) and a closed (ON) state. By controlling the switching equilibrium constant (KS) of such structure-switching templates, we can finely program their transcription efficiency. We have derived a simplified kinetic model that describes our experimental data with high accuracy, supporting the predictability of our approach. Similarly, we have also performed different molecular dynamic simulations that provide an atomistic description of our results. Finally, the introduction of such a conformational change mechanism in target-responsive templates allows rational control of their dynamic range (i.e., the range of target concentration to which they respond) and of their input-output behavior. The structure-switching templates demonstrated in this work are particularly easy to design and provide a highly programmable approach to control cell-free transcription, a feature that can be of great utility in fields like sensing and RNA therapeutics.