Programming cell-free biosensors with DNA strand displacement circuits

TLDR

Cell‑free biosensors provide powerful platforms for monitoring human and environmental health. The study expands cell‑free biosensor capabilities by interfacing them with toehold‑mediated strand displacement circuits and develops design rules for integrating the ROSALIND small‑molecule sensor with hybrid RNA–DNA circuits to fine‑tune reaction kinetics. Using these design rules, the authors built 12 hybrid RNA–DNA circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND). They demonstrate an analog‑to‑digital converter circuit that generates binary outputs encoding the detected molecule’s concentration range, illustrating a pathway to smart diagnostics that leverage molecular computation to improve biosensor speed and utility.

Abstract

Abstract Cell-free biosensors are powerful platforms for monitoring human and environmental health. Here, we expand their capabilities by interfacing them with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing a small molecule sensing platform called ROSALIND with toehold-mediated strand displacement to construct hybrid RNA–DNA circuits that allow fine-tuning of reaction kinetics. We use these design rules to build 12 different circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND). Finally, we demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the molecule being detected. We believe this work establishes a pathway to create ‘smart’ diagnostics that use molecular computations to enhance the speed and utility of biosensors.