Cell-free biosensors are emerging as powerful platforms for monitoring human and environmental health. Here, we expand the capabilities of biosensors by interfacing their outputs with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. This platform consists of T7 RNA polymerase, an allosteric transcription factor and a DNA template that together regulate the synthesis of an invading RNA strand that can activate fluorescence from a DNA signal gate – a double-stranded DNA consisting of a quencher strand and a fluorophore strand with a toehold region. We first show that the design of the DNA signal gate can be optimized to make in vitro transcription compatible with DNA strand displacement. Next, we systematically develop design principles for optimizing the secondary structure of the synthesized RNA to tune the kinetics of strand displacement, notably improving the biosensing response speed. We also apply this principle to interface strand displacement circuits with several different transcription factors to create biosensors for their cognate ligands. Finally, we address a current limitation of cell-free biosensors by using a model-driven approach to design and build a multi-layer strand displacement circuit that acts like an analog-to-digital converter to provide information about the concentration range of the target molecule being sensed. Supplementary data file 2 includes all unprocessed, uncropped polyacrylamide gel images shown in the manuscript and supplementary data file 3 includes all source data presented in this study generated by plate reader measurements, Qubit measurements and Fiji-ImageJ analysis. Supplementary data file 2 is provided as a zip file with each image annotated with a text file. All gel images shown in this file are urea-PAGE-TBE-gels that were stained with SYBR gold and imaged using a BioRad ChemiDoc imaging system.
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Please refer to the Materials and Methods section of the manuscript for reproducing the data presented in this dataset.