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.
Highlights
Cell-free biosensors are powerful platforms for monitoring human and environmental health
To interface ROSALIND with toehold-mediated DNA strand displacement (TMSD), we first sought to validate that a single-stranded RNA can strand-displace a DNA signal gate
We discovered that varying the T7 RNA polymerase (RNAP) transcription efficiency of each DNA template[36] contributes to this discrepancy, RNA secondary structure has a greater impact on the TMSD response speed (Extended Data Fig. 2)
Summary
Cell-free biosensors are powerful platforms for monitoring human and environmental health 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). Cell-free biosensing is emerging as a low-cost, easy-to-use and field-deployable diagnostic technology platform that can detect a range of chemical compounds related to human and environmental health[1,2] At their core, these systems consist of two layers: an RNA or protein-based biosensing layer and a reporter construct output layer. There is a great potential for TMSD-based information processing to improve cell-free biosensors
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