Programming Cell-Free Biosensors with DNA Strand Displacement Circuits

Abstract

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. We develop design rules for interfacing biosensors with strand displacement circuits, show that these circuits allow fine-tuning of reaction kinetics and faster response times, and 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 target molecule being detected. We believe this work establishes a pathway to create 'smart' diagnostics that use molecular computations to enhance the speed, robustness and utility of biosensors.

Building an RNA-Based Toggle Switch Using Inhibitory RNA Aptamers

“…On the basis of previous works on minimalist RNA-based circuits 28 and theoretical modeling of an RNA-based toggle switch, 32 we demonstrated the behavior of this specific network in vitro and highlighted the potential for implementing this network in living cells and other in vitro applications. 20 , 21 Degrading conditions are closer to how the circuit would behave in a cellular environment and can help to understand its performance in that context. The next step would be testing the RNA-based toggle switch in more cell-like conditions such as cell lysates.…”

“…In vitro transcription systems have become an attractive alternative not only to construct and characterize biological circuits without involving the complexities of living systems, but also for other applications such as diagnosis. 20,21 With full control over the concentrations and stoichiometries of each component, these platforms are ideal for the rapid prototyping of genetic circuit designs as well as for developing mathematical models to characterize these systems and help fine-tune their design. 22,23 The most commonly used enzymes for in vitro transcription systems are bacteriophage RNA polymerases due to their high processivity for catalyzing the formation of RNA from DNA templates.…”

Co-transcriptional RNA strand displacement circuits

“…Circuits could then respond multiple times to changing input signals, overcoming a current limitation in DNA computing (11,19). Additionally, regulating input production with allosteric transcription factors allows ctRSD circuits that process non-nucleic acid inputs to be readily developed for smart diagnostics (30,31). Finally, the ability to transcriptionally encode strand displacement components in DNA plasmids allows nucleic acid computing to be employed in a number of new environments where DNA computing is limited due to degradation ( 16), e.g.…”

“…Further, ctRSD circuits are designed so that state-of-the-art DNA-based circuits capable of neural network computations and pattern recognition (4, 6) could be directly adopted. ctRSD should enable the power of TMSD circuits to be realized in biological systems for smart diagnostics or sensors (6,30,31). Ultimately, ctRSD circuits could be genetically encoded and continuously operated inside living cells.…”