A nanopore interface for high bandwidth DNA computing

DNA has emerged as a powerful substrate for programming information processing machines at the nanoscale. Among the DNA computing primitives used today, DNA strand displacement (DSD) is arguably the most popular, with DSD-based circuit applications ranging from disease diagnostics to molecular artificial neural networks. The outputs of DSD circuits are generally read using fluorescence spectroscopy. However, due to the spectral overlap of typical small-molecule fluorescent reporters, the number of unique outputs that can be detected in parallel is limited, requiring complex optical setups or spatial isolation of reactions to make output bandwidths scalable. Here, we present a multiplexable sequencing-free readout method that enables real-time, kinetic measurement of DSD circuit activity through highly parallel, direct detection of barcoded output strands using nanopore sensor array technology (Oxford Nanopore Technologies' MinION device). We show that engineered reporter probes can be detected and classified with high accuracy at the single-molecule level directly from raw nanopore signals using deep learning. We then demonstrate this method's utility in multiplexed detection of clinically relevant microRNA sequences. These results increase DSD output bandwidth by an order of magnitude over what is possible with fluorescence spectroscopy, laying the foundations for a new paradigm in DNA circuit readout and programmable multiplexed molecular diagnostics using portable nanopore devices.

Nonenzymatic DNA-Based Fluorescence Biosensor Combining Carbon Dots and Graphene Oxide with Target-Induced DNA Strand Displacement for microRNA Detection

Based on a fluorescence “on-off-on” strategy, we fabricated a simple and highly sensitive DNA-based fluorescence biosensor for the detection of micro (mi)RNA from carbon dots (CDs) and graphene oxide (GO) without complicated and time-consuming operations. CDs were successfully synthesized and conjugated to the end of a single-stranded fuel DNA that was adsorbed onto the surface of GO through π-π stacking, resulting in fluorescence quenching. In the presence of the target miRNA let-7a, the fuel DNA was desorbed from the GO surface, and fluorescence was restored through two successive toehold-mediated strand displacement reactions on double-stranded DNA-modified gold nanoparticles. The target miRNA let-7a was recycled, leading to signal amplification. The concentration of let-7a was proportional to the degree of fluorescence recovery. Under optimal conditions, there was a good linear relationship between the relative fluorescence intensity and let-7a concentration in the range of 0.01–1 nM, with a detection limit of 7.8 pM. With its advantages of signal amplification and high biocompatibility, this fluorescence sensing strategy can be applied to the detection of a variety of target miRNAs and can guide the design of novel biosensors with improved properties.

Autonomous DNA nanostructures instructed by hierarchically concatenated chemical reaction networks

Concatenation and communication between chemically distinct chemical reaction networks (CRNs) is an essential principle in biology for controlling dynamics of hierarchical structures. Here, to provide a model system for such biological systems, we demonstrate autonomous lifecycles of DNA nanotubes (DNTs) by two concatenated CRNs using different thermodynamic principles: (1) ATP-powered ligation/restriction of DNA components and (2) input strand-mediated DNA strand displacement (DSD) using energy gains provided in DNA toeholds. This allows to achieve hierarchical non-equilibrium systems by concurrent ATP-powered ligation-induced DSD for activating DNT self-assembly and restriction-induced backward DSD reactions for triggering DNT degradation. We introduce indirect and direct activation of DNT self-assemblies, and orthogonal molecular recognition allows ATP-fueled self-sorting of transient multicomponent DNTs. Coupling ATP dissipation to DNA nanostructures via programmable DSD is a generic concept which should be widely applicable to organize other DNA nanostructures, and enable the design of automatons and life-like systems of higher structural complexity.

DNA breathing initiated strand displacement circuits for mimicking ant foraging with nanopore readout

DNA strand displacement reaction is essential for the development of molecular computing based on DNA nanotechnology. Additional DNA strand exchange strategies with high selectivity for input will enable novel complex systems including biosensing applications. Most approaches use bulk readout methods based on fluorescent probes that complicate the monitoring of parallel computations. Herein we propose an autocatalytic strand displacement (ACSD) circuit, which is initiated by DNA breathing and accelerated by seesaw catalytic reaction. The special initiation mechanism of the ACSD circuit enables detection of single base mutations at multiple sites in the input strand with much higher sensitivity than classic toehold-mediated strand displacement. A swarm intelligence model is constructed using the ACSD circuit to mimic foraging behaviour of ants. We introduce a multiplexed nanopore sensing platform to report the output results of a parallel path selection system on the single-molecule level. The ACSD strategy and nanopore multiplexed readout method enhance the toolbox for the future development of DNA computing.

Using Threshold DNA Strand Displacement to Construct a Half Adder

DNA strand displacement has the advantages of product predictability, programmability and threshold, and it is often used to build DNA-based logic operation systems. In this paper, we use DNA strand displacement to have different reaction priorities in different length ranges of the toehold domain to form the effect of the threshold and construct the logical AND gate and XOR gate. Logical operations use single-stranded DNA as the input signal, and the brightness of the fluorescence is used to measure the results. Then, the logic AND gate and XOR gate are used as basic logic units to form a half adder in parallel. Finally, we use Visual DSD to simulate and analyze the logic AND gate, XOR gate and half adder. The simulation results show that the logic gates constructed in this paper have good theoretical feasibility and effectiveness. This work provides a potential design idea for DNA-based arithmetic function operations and more advanced logic operations.