Tune, Extend, and Narrow the Useful Dynamic Range of Cell‐Free Transcription Biosensors Through Programmable DNA‐Based Stem‐Loop Hairpin Reporters

ABSTRACT In this work, we present a general, modular strategy to tune, extend, and narrow the dynamic range of cell‐free transcription biosensing platforms by integrating programmable, structure‐switching DNA stem‐loop reporters into in vitro transcription (IVT) circuits. To do so, we engineered a set of stem‐loop DNA reporters whose dynamic range for detecting a specific RNA output can be precisely controlled by adjusting their switching equilibrium constant ( K S ). This straightforward approach enables the dynamic range of a model cell‐free transcription biosensor to be programmed across more than two orders of magnitude (observed affinity K D obs from 0.16 ± 0.02 nM up to 23 ± 4 nM). By combining DNA‐based reporters with differing affinities, we further expanded the dynamic range of a cell‐free transcription biosensor well beyond the conventional two orders of magnitude, achieving up to 10 4 ‐fold coverage. We also demonstrate two‐step dynamic responses by mixing hairpin reporters with highly distinct affinities. Finally, integration of signaling and non‐signaling stem‐loop reporters allowed us to compress the dynamic range of a model transcription biosensor to as little as three‐fold enabling heightened sensitivity. Overall, this modular framework enables the customization of cell‐free transcription biosensor sensitivity and response profiles, overcoming key limitations inherent to single‐site transcriptional reporter designs.