A survey on molecular-scale learning systems with relevance to DNA computing.

DNA computing has emerged as a promising alternative to achieve programmable behaviors in chemistry by repurposing the nucleic acid molecules into chemical hardware upon which synthetic chemical programs can be executed. These chemical programs are capable of simulating diverse behaviors, including boolean logic computation, oscillations, and nanorobotics. Chemical environments such as the cell are marked by uncertainty and are prone to random fluctuations. For this reason, potential DNA-based molecular devices that aim to be deployed into such environments should be capable of adapting to the stochasticity inherent in them. In keeping with this goal, a new subfield has emerged within DNA computing, focusing on developing approaches that embed learning and inference into chemical reaction systems. If realized in biochemical contexts, such molecular machines can engender novel applications in fields such as biotechnology, synthetic biology, and medicine. Therefore, it would be beneficial to review how different ideas were conceived, how the progress has been so far, and what the emerging ideas are in this nascent field of 'molecular-scale learning'.

A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens.

Access to fast and reliable nucleic acid testing continues to play a key role in controlling the COVID-19 pandemic, especially in the context of increased vaccine break-through risks due to new variants. We report a rapid, low-cost (~ 2 USD), simple-to-use nucleic acid test kit for self-administered at-home testing without lab instrumentation. The entire sample-to-answer workflow takes < 60 min, including noninvasive sample collection, one-step RNA preparation, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in a thermos, and direct visual inspection of a colorimetric test result. To facilitate long-term storage without cold-chain, a fast one-pot lyophilization protocol was developed to preserve all required biochemical reagents of the colorimetric RT-LAMP test in a single microtube. Notably, the lyophilized RT-LAMP assay demonstrated reduced false positives as well as enhanced tolerance to a wider range of incubation temperatures compared to solution-based RT-LAMP reactions. We validated our RT-LAMP assay using simulated infected samples, and detected a panel of SARS-CoV-2 variants with successful detection of all variants that were available to us at the time. With a simple change of the primer set, our lyophilized RT-LAMP home test can be easily adapted as a low-cost surveillance platform for other pathogens and infectious diseases of global public health importance.

Design and construction of double-decker tile as a route to three-dimensional periodic assembly of DNA.

DNA is a useful material for nanoscale construction. Due to highly specific Watson-Crick base pairing, the DNA sequences can be designed to form small tiles or origami. Adjacent helices in such nanostructures are connected via Holliday junction-like crossovers. DNA tiles can have sticky ends which can then be programmed to form large one-dimensional and two-dimensional periodic lattices. Recently, a three-dimensional DNA lattice has also been constructed. Here we report the design and construction of a novel DNA cross tile, called the double-decker tile. Its arms are symmetric and have four double helices each. Using its sticky ends, large two-dimensional square lattices have been constructed which are on the order of tens of micrometers. Furthermore, it is proposed that the sticky ends of the double-decker tile can be programmed to form a three-dimensional periodic lattice with large cavities that could be used as a scaffold for precise positioning of molecules in space.

On robotic optimal path planning in polygonal regions with pseudo-Euclidean metrics.

This paper presents several results on some cost-minimizing path problems in polygonal regions. For these types of problems, an approach often used to compute approximate optimal paths is to apply a discrete search algorithm to a graph G(epsilon) constructed from a discretization of the problem; this graph is guaranteed to contain an epsilon-good approximate optimal path, i.e., a path with a cost within (1 + epsilon) factor of that of an optimal path, between given source and destination points. Here, epsilon > 0 is the user-defined error tolerance ratio. We introduce a class of piecewise pseudo-Euclidean optimal path problems that includes several non-Euclidean optimal path problems previously studied and show that the BUSHWHACK algorithm, which was formerly designed for the weighted region optimal path problem, can be generalized to solve any optimal path problem of this class. We also introduce an empirical method called the adaptive discretization method that improves the performance of the approximation algorithms by placing discretization points densely only in areas that may contain optimal paths. It proceeds in multiple iterations, and in each iteration, it varies the approximation parameters and fine tunes the discretization.

An autonomously self-assembling dendritic DNA nanostructure for target DNA detection.

There is a growing need for sensitive and reliable nucleic acid detection methods that are convenient and inexpensive. Responsive and programmable DNA nanostructures have shown great promise as chemical detection systems. Here, we describe a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. The detection protocol is executed autonomously without external intervention. Detection begins when a specific, single-stranded target DNA strand (T) triggers a hybridization chain reaction (HCR) between two, distinct DNA hairpins (α and ß). Each hairpin opens and hybridizes up to two copies of the other. In the absence of T, α and ß are stable and remain in their poised, closed-hairpin form. In the presence of T, α hairpins are opened by toe-hold mediated strand-displacement, each of which then opens and hybridizes two ß hairpins. Likewise, each opened ß hairpin can open and hybridize two α hairpins. Hence, each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a high molecular weight nanostructure. This HCR system has minimal sequence constraints, allowing reconfiguration for the detection of arbitrary target sequences. Here, we demonstrate detection of unique sequence identifiers of HIV and Chlamydia pathogens.

A DNA nanotransport device powered by polymerase phi29.

Polymerases are a family of enzymes responsible for copying or replication of nucleic acids (DNA or RNA) templates and hence sustenance of life processes. In this paper, we present a method to exploit a strand-displacing polymerase phi29 as a driving force for nanoscale transportation devices. The principal idea behind the device is strong strand displacement ability of phi29, which can displace any DNA strand from its template while extending a primer hybridized to the template. This capability of phi29 is used to power the movement of a target nanostructure on a DNA track. The major advantage of using a polymerase driven nanotransportation device as compared to other existing nanorobotical devices is its speed. phi29 polymerase can travel at the rate of 2000 nucleotides per minute at room temperature, which translates to approximately 680 nm min(-1) on a nanostructure. We also demonstrate transportation of a DNA cargo on a DNA track with the help of fluorescence resonance electron transfer data.

Programming DNA tube circumferences.

Synthesizing molecular tubes with monodisperse, programmable circumferences is an important goal shared by nanotechnology, materials science, and supermolecular chemistry. We program molecular tube circumferences by specifying the complementarity relationships between modular domains in a 42-base single-stranded DNA motif. Single-step annealing results in the self-assembly of long tubes displaying monodisperse circumferences of 4, 5, 6, 7, 8, 10, or 20 DNA helices.

Programmable DNA self-assemblies for nanoscale organization of ligands and proteins.

We demonstrate the precise control of periodic spacing between individual protein molecules by programming the self-assembly of DNA tile templates. In particular, we report the application of two self-assembled periodic DNA structures, two-dimensional nanogrids, and one-dimensional nanotrack, as template for programmable self-assembly of streptavidin protein arrays with controlled density.

Three-helix bundle DNA tiles self-assemble into 2D lattice or 1D templates for silver nanowires.

We present a DNA nanostructure, the three-helix bundle (3HB), which consists of three double helical DNA domains connected by six immobile crossover junctions such that the helix axes are not coplanar. The 3HB motif presents a triangular cross-section with one helix lying in the groove formed by the other two. By differential programming of sticky-ends, 3HB tiles can be arrayed in two distinct lattice conformations: one-dimensional filaments and two-dimensional lattices. Filaments and lattices have been visualized by high-resolution, tapping mode atomic force microscopy (AFM) under buffer. Their dimensions are shown to be in excellent agreement with designed structures. We also demonstrate an electroless chemical deposition for fabricating metallic nanowires templated on self-assembled filaments. The metallized nanowires have diameters down to 20 nm and display Ohmic current-voltage characteristic.

Parallel molecular computations of pairwise exclusive-or (XOR) using DNA "string tile" self-assembly.

Self-assembling DNA nanostructures are an efficient means of executing parallel molecular computations. However, previous experimental demonstrations of computations by DNA tile self-assembly only allowed for one set of distinct input to be processed at a time. Here, we report the multibit, parallel computation of pairwise exclusive-or (XOR) using DNA "string tile" self-assembly. A set of DNA tiles encoding the truth table for the XOR logical operation was constructed. Parallel tile self-assembly and ligation led to the formation of reporter DNA strands which encoded both the input and the output of the computations. These reporter strands provided a molecular look-up table containing all possible pairwise XOR calculations up to a certain input size. The computation was readout by sequencing the cloned reporter strands. This is the first experimental demonstration of a parallel computation by DNA tile self-assembly in which a large number of distinct input were simultaneously processed.

DNA-templated self-assembly of protein and nanoparticle linear arrays.

Self-assembling DNA tiling lattices represent a versatile system for nanoscale construction. Self-assembled DNA arrays provide an excellent template for spatially positioning other molecules with increased relative precision and programmability. Here we report an experiment using a linear array of DNA triple crossover tiles to controllably template the self-assembly of single-layer or double-layer linear arrays of streptavidin molecules and streptavidin-conjugated nanogold particles through biotin-streptavidin interaction. The organization of streptavidin and its conjugated gold nanoparticles into periodic arrays was visualized by atomic force microscopy and scanning electron microscopy.

DNA-templated self-assembly of protein arrays and highly conductive nanowires.

A DNA nanostructure consisting of four four-arm junctions oriented with a square aspect ratio was designed and constructed. Programmable self-assembly of 4 x 4 tiles resulted in two distinct lattice morphologies: uniform-width nanoribbons and two-dimensional nanogrids, which both display periodic square cavities. Periodic protein arrays were achieved by templated self-assembly of streptavidin onto the DNA nanogrids containing biotinylated oligonucleotides. On the basis of a two-step metallization procedure, the 4 x 4 nanoribbons acted as an excellent scaffold for the production of highly conductive, uniform-width, silver nanowires.

DNA nanotubes self-assembled from triple-crossover tiles as templates for conductive nanowires.

DNA-based nanotechnology is currently being developed as a general assembly method for nanopatterned materials that may find use in electronics, sensors, medicine, and many other fields. Here we present results on the construction and characterization of DNA nanotubes, a self-assembling superstructure composed of DNA tiles. Triple-crossover tiles modified with thiol-containing double-stranded DNA stems projected out of the tile plane were used as the basic building blocks. Triple-crossover nanotubes display a constant diameter of approximately 25 nm and have been observed with lengths up to 20 microm. We present high-resolution images of the constructs, experimental evidence of their tube-like nature as well as data on metallization of the nanotubes to form nanowires, and electrical conductivity measurements through the nanowires. DNA nanotubes represent a potential breakthrough in the self-assembly of nanometer-scale circuits for electronics layout because they can be targeted to connect at specific locations on larger-scale structures and can subsequently be metallized to form nanometer-scale wires. The dimensions of these nanotubes are also perfectly suited for applications involving interconnection of molecular-scale devices with macroscale components fabricated by conventional photolithographic methods.

Directed nucleation assembly of DNA tile complexes for barcode-patterned lattices.

The programmed self-assembly of patterned aperiodic molecular structures is a major challenge in nanotechnology and has numerous potential applications for nanofabrication of complex structures and useful devices. Here we report the construction of an aperiodic patterned DNA lattice (barcode lattice) by a self-assembly process of directed nucleation of DNA tiles around a scaffold DNA strand. The input DNA scaffold strand, constructed by ligation of shorter synthetic oligonucleotides, provides layers of the DNA lattice with barcode patterning information represented by the presence or absence of DNA hairpin loops protruding out of the lattice plane. Self-assembly of multiple DNA tiles around the scaffold strand was shown to result in a patterned lattice containing barcode information of 01101. We have also demonstrated the reprogramming of the system to another patterning. An inverted barcode pattern of 10010 was achieved by modifying the scaffold strands and one of the strands composing each tile. A ribbon lattice, consisting of repetitions of the barcode pattern with expected periodicity, was also constructed by the addition of sticky ends. The patterning of both classes of lattices was clearly observable via atomic force microscopy. These results represent a step toward implementation of a visual readout system capable of converting information encoded on a 1D DNA strand into a 2D form readable by advanced microscopic techniques. A functioning visual output method would not only increase the readout speed of DNA-based computers, but may also find use in other sequence identification techniques such as mutation or allele mapping.