Allosteric transcription factors (aTFs) naturally create this interface by activating transcription of a programmable RNA sequence upon detection of a chemical target. We therefore sought to create an interface that can convert the binding event of a chemical target to changes in nucleic acid strands that can trigger TMSD cascades. Thus, there is a great potential for TMSD-based information processing to improve cell-free biosensors.Īlthough TMSD circuits have been used to detect nucleic acid targets such as microRNAs 24, 25 and human pathogens 26, there are currently no general design rules for triggering TMSD circuits with small molecules to enable their use in cell-free biosensors. TMSD has led to the development of powerful devices including in vitro oscillators 18, catalytic amplifiers 19, autonomous molecular motors 20, 21 and reprogrammable DNA nanostructures 22, 23. In addition, reaction kinetics can be precisely tuned by changing the strength of the ‘toeholds’-single-stranded regions within the DNA gates that initiate the strand displacement process 17. The well-characterized thermodynamics of DNA base pairing enable large networks to be built from simple building blocks.
By configuring DNA gates into different network architectures, a range of operations can be performed such as signal restoration 12, signal amplification 13 and logic computation 14, 15, much like a general chemical computational architecture 16. In TMSD, single-stranded DNA (ssDNA) inputs exchange strands with double-stranded DNA ‘gates’ via complementary base pairing interactions to produce ssDNA output strands. Here, we develop a generalizable information processing layer to enhance and expand the function of ROSALIND by leveraging toehold-mediated DNA strand displacement (TMSD)-a computationally powerful DNA nanotechnology that can process molecular information in vitro 11. However, these circuits still directly act on either the sensing or the output layer, limiting our ability to improve and expand their function. Similarly, we have previously shown that RNA-based circuits can be added to a cell-free biosensors platform called RNA Output Sensors Activated by Ligand INDuction (ROSALIND) to improve their specificity and sensitivity without engineering the protein biosensors 7. For this reason, genetic information processing layers that implement logic and feedback have been extensively leveraged and engineered in synthetic cellular systems 9, 10. Such information processing layers are a natural feature of organisms and enable cells to activate stress responses, guide development and make behavioral decisions on the basis of intracellular and extracellular cues 8. However, existing cell-free biosensors often lack an information processing layer that can manipulate responses from the sensing layer before signal generation (Fig. We believe this work establishes a pathway to create ‘smart’ diagnostics that use molecular computations to enhance the speed and utility of biosensors. 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 use these design rules to build 12 different circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND).
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.
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. Cell-free biosensors are powerful platforms for monitoring human and environmental health.
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