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

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 (KS). 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 KDobs 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 104-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.