In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA–DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5′ or 3′ end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5′ end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

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In dynamic nucleic acids nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand which binds to a toehold, a single-stranded region on the duplex. With proper design and kinetic control, strand displacement is used to perform logic operations on molecular level to trigger the conformational change in nanostructures, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA, there has not been a comparable systematic work done for RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand) and mismatches influence the strand displacement kinetics. Through comparing the reaction rates, combined with previous theoretical studies, we observed reaction acceleration with increasing toehold length and placement of toehold at 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. Comparison of RNA displacement and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is in part explainable by the thermodynamic stabilities of the respective toehold regions, but also suggest that the rearrangement from B-form to A-form helix in case of RNA invading DNA might play a role in the kinetics. The measured kinetics of toehold-mediated strand displacement will be important in understanding and construction of more complex dynamic nucleic acid systems.

Gene expression has great potential to be used as a clinical diagnostic tool. Researchers have discovered a wealth of patterns in gene expression that are predictive of a wide range of conditions, from liver disease to infectious disease, oncological relapse risk to stratifying autoimmune patients. Despite the progress in identifying these gene expression signatures, clinical translation has been hampered by a lack of purpose-built, readily deployable testing platforms. Deploying these tests to the clinic has relied on expensive, specialized machines, like sequencers, or laborious PCR protocols that require running a dozen or more reactions in parallel. We have developed Competitive Amplification Networks (CANs) to enable analysis of an entire gene expression signature in a single PCR reaction. CANs consist of natural and synthetic amplicons that compete for shared primers during amplification, forming a reaction network that leverages the molecular machinery of PCR. These reaction components are tuned such that the final fluorescent signal from the assay is exactly calibrated to the conclusion of a statistical model. In essence, the reaction acts as a biological computer, simultaneously detecting the RNA targets, interpreting their level in the context of the gene expression signature, and aggregating their contributions to the final diagnosis. We demonstrate the clinical validity of this technique by designing a CAN around a gene expression signature for discriminating between fevers of viral origin and bacterial origin. When tested against twenty patient blood samples, our assay achieved perfect diagnostic agreement with the gold-standard approach of measuring each gene independently. At the same time, the CAN assay was faster and easier to use. Crucially, CAN assays are compatible with existing qPCR instruments and workflows. CANs hold the potential to enable rapid deployment and massive scalability of gene expression analysis to clinical laboratories around the world, in highly developed and low-resource settings alike.

Dynamic DNA nanotechnology involves the use of DNA strands to create programmable reaction networks and nanodevices. The key reaction in dynamic DNA nanotechnology is the exchange of DNA strands between different molecular species, which is achieved through three‐way and four‐ way DNA exchange reactions. While both of these reactions have been widely used to build reaction circuits, the four‐way exchange reaction has traditionally been slower and less efficient than the three‐way reaction. In this paper, we describe a new mechanism to optimise the kinetics of the four‐ way DNA exchange reaction by adding bulges to the toeholds of the four‐way DNA complexes involved in the reaction. These bulges facilitate an alternative branch migration mechanism and destabilise the four‐way DNA junction, increasing the branch migration rate and unbinding rate of the four‐way exchange reaction, bringing it closer to the kinetics of the three‐way reaction. This new mechanism has the potential to expand the field of dynamic DNA nanotechnology by enabling efficient four‐way DNA exchange reactions for in vivo applications.