Pioneering artificial cell-like structures with DNA nanotechnology-based liquid-liquid phase separation.

Recent studies have revealed that liquid-liquid phase separation (LLPS) plays crucial roles in various cellular functions. Droplets formed via LLPS within cells, often referred to as membraneless organelles, serve to concentrate specific molecules, thus enhancing biochemical reactions. Artificial LLPS systems have been utilized to construct synthetic cell models, employing a range of synthetic molecules. LLPS systems based on DNA nanotechnology are particularly notable for their designable characteristics in droplet formation, dynamics, properties, and functionalities. This review surveys recent advancements in DNA-based LLPS systems, underscoring the programmability afforded by DNA's base-pair specific interactions. We discuss the fundamentals of DNA droplet formation, including temperature-dependence and physical properties, along with the precise control achievable through sequence design. Attention is given to the phase separation of DNA nanostructures on two-dimensional closed interfaces, which results in spatial pattern formation at the interface. Furthermore, we spotlight the potential of DNA droplet computing for cancer diagnostics through specific microRNA pattern recognition. We envision that DNA-based LLPS presents a versatile platform for the exploration of cellular mimicry and opens innovative ways for the development of functional synthetic cells.

H-ACO with Consecutive Bases Pairing Constraint for Designing DNA Sequences.

DNA computing is a novel computing method that does not rely on traditional computers. The design of DNA sequences is a crucial step in DNA computing, and the quality of the sequence design directly affects the results of DNA computing. In this paper, a new constraint called the consecutive base pairing constraint is proposed to limit specific base pairings in DNA sequence design. Additionally, to improve the efficiency and capability of DNA sequence design, the Hierarchy-ant colony (H-ACO) algorithm is introduced, which combines the features of multiple algorithms and optimizes discrete numerical calculations. Experimental results show that the H-ACO algorithm performs well in DNA sequence design. Finally, this paper compares a series of constraint values and NUPACK simulation data with previous design results, and the DNA sequence set designed in this paper has more advantages.

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

DNA Logic Gates Integrated on DNA Substrates in Molecular Computing.

Due to nucleic acid's programmability, it is possible to realize DNA structures with computing functions, and thus a new generation of molecular computers is evolving to solve biological and medical problems. Pioneered by Milan Stojanovic, Boolean DNA logic gates created the foundation for the development of DNA computers. Similar to electronic computers, the field is evolving towards integrating DNA logic gates and circuits by positioning them on substrates to increase circuit density and minimize gate distance and undesired crosstalk. In this minireview, we summarize recent developments in the integration of DNA logic gates into circuits localized on DNA substrates. This approach of all-DNA integrated circuits (DNA ICs) offers the advantages of biocompatibility, increased circuit response, increased circuit density, reduced unit concentration, facilitated circuit isolation, and facilitated cell uptake. DNA ICs can face similar challenges as their equivalent circuits operating in bulk solution (bulk circuits), and new physical challenges inherent in spatial localization. We discuss possible avenues to overcome these obstacles.

Programming and monitoring surface-confined DNA computing.

DNA-based molecular computing has evolved to encompass a diverse range of functions, demonstrating substantial promise for both highly parallel computing and various biomedical applications. Recent advances in DNA computing systems based on surface reactions have demonstrated improved levels of specificity and computational speed compared to their solution-based counterparts that depend on three-dimensional molecular collisions. Herein, computational biomolecular interactions confined by various surfaces such as DNA origamis, nanoparticles, lipid membranes and chips are systematically reviewed, along with their manipulation methodologies. Monitoring techniques and applications for these surface-based computing systems are also described. The advantages and challenges of surface-confined DNA computing are discussed.

Programmable Molecular Signal Transmission Architecture and Reactant Regeneration Strategy Driven by EXO λ for DNA Circuits.

The characteristics of DNA hybridization enable molecular computing through strand displacement reactions, facilitating the construction of complex DNA circuits, which is an important way to realize information interaction and processing at a molecular level. However, signal attenuation in the cascade and shunt process hinders the reliability of the calculation results and further expansion of the DNA circuit scale. Here, we demonstrate a novel programmable exonuclease-assisted signal transmission architecture, where DNA strand with toehold employed to inhibit the hydrolysis process of EXO λ is applied in DNA circuits. We construct a series circuit with variable resistance and a parallel circuit with constant current source, ensuring excellent orthogonal properties between input and output sequences while maintaining low leakage (<5%) during the reaction. Additionally, a simple and flexible exonuclease-driven reactant regeneration (EDRR) strategy is proposed and applied to construct parallel circuits with constant voltage sources that could amplify the output signal without extra DNA fuel strands or energy. Furthermore, we demonstrate the effectiveness of the EDRR strategy in reducing signal attenuation during cascade and shunt processes by constructing a four-node DNA circuit. These findings offer a new approach to enhance the reliability of molecular computing systems and expand the scale of DNA circuits in the future.

Implementing Logic Gates by DNA Crystal Engineering.

DNA self-assembly computation is attractive for its potential to perform massively parallel information processing at the molecular level while at the same time maintaining its natural biocompatibility. It has been extensively studied at the individual molecule level, but not as much as ensembles in 3D. Here, the feasibility of implementing logic gates, the basic computation operations, in large ensembles: macroscopic, engineered 3D DNA crystals is demonstrated. The building blocks are the recently developed DNA double crossover-like (DXL) motifs. They can associate with each other via sticky-end cohesion. Common logic gates are realized by encoding the inputs within the sticky ends of the motifs. The outputs are demonstrated through the formation of macroscopic crystals that can be easily observed. This study points to a new direction of construction of complex 3D crystal architectures and DNA-based biosensors with easy readouts.

DNA-based customized functional modules for signal transformation.

Information on the temporal and spatial scale of cellular molecules in biological systems is crucial for estimating life processes and may be conducive to an improved understanding of disease progression. This intracellular and extracellular information is often difficult to obtain at the same time due to the limitations of accessibility and sensing throughput. DNA is an excellent material for in vivo and in vitro applications, and can be used to build functional modules that can transform bio-information (input) into ATCG sequence information (output). Due to their small volume and highly amenable programming, DNA-based functional modules provide an opportunity to monitor a range of information, from transient molecular events to dynamic biological processes. Over the past two decades, with the advent of customized strategies, a series of functional modules based on DNA networks have been designed to gather different information about molecules, including the identity, concentration, order, duration, location, and potential interactions; the action of these modules are based on the principle of kinetics or thermodynamics. This paper summarizes the available DNA-based functional modules that can be used for biomolecular signal sensing and transformation, reviews the available designs and applications of these modules, and assesses current challenges and prospects.

DNA strand displacement based computational systems and their applications.

DNA computing has become the focus of computing research due to its excellent parallel processing capability, data storage capacity, and low energy consumption characteristics. DNA computational units can be precisely programmed through the sequence specificity and base pair principle. Then, computational units can be cascaded and integrated to form large DNA computing systems. Among them, DNA strand displacement (DSD) is the simplest but most efficient method for constructing DNA computing systems. The inputs and outputs of DSD are signal strands that can be transferred to the next unit. DSD has been used to construct logic gates, integrated circuits, artificial neural networks, etc. This review introduced the recent development of DSD-based computational systems and their applications. Some DSD-related tools and issues are also discussed.

Design and Simulation of an Autonomous Molecular Mechanism Using Spatially Localized DNA Computation.

As a well-established technique, DNA synthesis offers interesting possibilities for designing multifunctional nanodevices. The micro-processing system of modern semiconductor circuits is dependent on strategies organized on silicon chips to achieve the speedy transmission of substances or information. Similarly, spatially localized structures allow for fixed DNA molecules in close proximity to each other during the synthesis of molecular circuits, thus providing a different strategy that of opening up a remarkable new area of inquiry for researchers. Herein, the Visual DSD (DNA strand displacement) modeling language was used to design and analyze the spatially organized DNA reaction network. The execution rules depend on the hybridization reaction caused by directional complementary nucleotide sequences. A series of DNA strand displacement calculations were organized on the locally coded travel track, and autonomous movement and addressing operations are gradually realized. The DNA nanodevice operates in this manner follows the embedded "molecular program", which improves the reusability and scalability of the same sequence domain in different contexts. Through the communication between various building blocks, the DNA device-carrying the target molecule moves in a controlled manner along the programmed track. In this way, a variety of molecular functional group transport and specific partition storage can be realized. The simulation results of the visual DSD tool provide qualitative and quantitative proof for the operation of the system.

Stochastic approximations of higher-molecular by bi-molecular reactions.

Reactions involving three or more reactants, called higher-molecular reactions, play an important role in mathematical modelling in systems and synthetic biology. In particular, such reactions underpin a variety of important bio-dynamical phenomena, such as multi-stability/multi-modality, oscillations, bifurcations, and noise-induced effects. However, as opposed to reactions involving at most two reactants, called bi-molecular reactions, higher-molecular reactions are biochemically improbable. To bridge the gap, in this paper we put forward an algorithm for systematically approximating arbitrary higher-molecular reactions with bi-molecular ones, while preserving the underlying stochastic dynamics. Properties of the algorithm and convergence are established via singular perturbation theory. The algorithm is applied to a variety of higher-molecular biochemical networks, and is shown to play an important role in synthetic biology.

DNA Droplets: Intelligent, Dynamic Fluid.

Breathtaking advances in DNA nanotechnology have established DNA as a promising biomaterial for the fabrication of programmable higher-order nano/microstructures. In the context of developing artificial cells and tissues, DNA droplets have emerged as a powerful platform for creating intelligent, dynamic cell-like machinery. DNA droplets are a microscale membrane-free coacervate of DNA formed through phase separation. This new type of DNA system couples dynamic fluid-like property with long-established DNA programmability. This hybrid nature offers an advantageous route to facile and robust control over the structures, functions, and behaviors of DNA droplets. This review begins by describing programmable DNA condensation, commenting on the physical properties and fabrication strategies of DNA hydrogels and droplets. By presenting an overview of the development pathways leading to DNA droplets, it is shown that DNA technology has evolved from static, rigid systems to soft, dynamic systems. Next, the basic characteristics of DNA droplets are described as intelligent, dynamic fluid by showcasing the latest examples highlighting their distinctive features related to sequence-specific interactions and programmable mechanical properties. Finally, this review discusses the potential and challenges of numerical modeling able to connect a robust link between individual sequences and macroscopic mechanical properties of DNA droplets.

DNA computing for gastric cancer analysis and functional classification.

Early identification of key biomarkers of malignant cancer is vital for patients' prognosis and therapies. There is research demonstrating that microRNAs are important biomarkers for cancer analysis. In this article, we used the DNA strand displacement mechanism (DSD) to construct the DNA computing system for cancer analysis. First, gene chips were obtained through bioinformatical training. These microRNA data and clinical traits were obtained from the Cancer Genome Atlas (TCGA) dataset. Second, we analyzed the expression data by using a weighted gene co-expression network (WGCNA) and found four biomarkers for two clinic features, respectively. Last, we constructed a DSD-based DNA computing system for cancer analysis. The inputs of the system are these identified biomarkers; the outputs are the fluorescent signals that represent their corresponding traits. The experiment and simulation results demonstrated the reliability of the DNA computing system. This DSD simulation system is lab-free but clinically meaningful. We expect this innovative method to be useful for rapid and accurate cancer diagnosis.

Graph Computation Using Algorithmic Self-Assembly of DNA Molecules.

DNA molecules have been used as novel computing tools, by which Synthetic DNA was designed to execute computing processes with a programmable sequence. Here, we proposed a parallel computing method using DNA origamis as agents to solve the three-color problem, an example of the graph problem. Each agent was fabricated with a DNA origami of ∼50 nm diameter and contained DNA probes with programmable sticky ends that execute preset computing processes. With the interaction of different nanoagents, DNA molecules self-assemble into spatial nanostructures, which embody the computation results of the three-color problem with polynomial numbers of computing nanoagents in a one-pot annealing step. The computing results were confirmed by atomic force microscopy. Our method is completely different from existing DNA computing methods in its computing algorithm, and it has an advantage in terms of computational complexity and results detection for solving graph problems.

Massively Parallel DNA Computing Based on Domino DNA Strand Displacement Logic Gates.

DNA computing has gained considerable attention due to the characteristics of high-density information storage and high parallel computing for solving computational problems. Building addressable logic gates with biomolecules is the basis for establishing biological computers. In the current calculation model, the multiinput AND operation often needs to be realized through a multilevel cascade between logic gates. Through experiments, it was found that the multilevel cascade causes signal leakage and affects the stability of the system. Using DNA strand displacement technology, we constructed a domino-like multiinput AND gate computing system instead of a cascade of operations, realizing multiinput AND computing on one logic gate and abandoning the traditional multilevel cascade of operations. Fluorescence experiments demonstrated that our methods significantly reduce system construction costs and improve the stability and robustness of the system. Finally, we proved stability and robustness of the domino AND gate by simulating the tic-tac-toe process with a massively parallel computing strategy.

Construction of Multiple Logic Circuits Based on Allosteric DNAzymes.

In DNA computing, the implementation of complex and stable logic operations in a universal system is a critical challenge. It is necessary to develop a system with complex logic functions based on a simple mechanism. Here, the strategy to control the secondary structure of assembled DNAzymes' conserved domain is adopted to regulate the activity of DNAzymes and avoid the generation of four-way junctions, and makes it possible to implement basic logic gates and their cascade circuits in the same system. In addition, the purpose of threshold control achieved by the allosteric secondary structure implements a three-input DNA voter with one-vote veto function. The scalability of the system can be remarkably improved by adjusting the threshold to implement a DNA voter with 2n + 1 inputs. The proposed strategy provides a feasible idea for constructing more complex DNA circuits and a highly integrated computing system.

Design and Realization of Triple dsDNA Nanocomputing Circuits in Microfluidic Chips.

DNA logic gates, nanocomputing circuits, have already implemented basic computations and shown great signal potential for nano logic material application. However, the reaction temperature and computing speed still limit its development. Performing complicated computations requires a more stable component and a better computing platform. We proposed a more stable design of logic gates based on a triple, double-stranded, DNA (T-dsDNA) structure. We demonstrated a half adder and a full adder using these DNA nanocircuits and performed the computations in a microfluidic chip device at room temperature. When the solutions were mixed in the device, we obtained the expected results in real time, which suggested that the T-dsDNA combined microfluidic chip provides a concise strategy for large DNA nanocircuits.

Sensitive Logic Nanodevices with Strong Response for Weak Inputs.

Sensitive sensing is critical when developing new calculation systems with weak input signals (ISs). In this work, a "weak-inputs-strong-outputs" strategy was proposed to guide the construction of sensitive logic nanodevices by coupling an input-induced reversible DNA computing platform with a hybridization chain reaction-based signal amplifier. By rational design of the sequence of computing elements (CEs) so as to avoid cross-talking between ISs and signal amplifier, the newly formed logic nanodevices have good sensitivity to the weak ISs even at low concentrations of CEs, and are able to perform YES, OR, NAND, NOR, INHIBIT, INHIBIT-OR and number classifier operation, showing that the DNA calculation proceeds in dilute solution medium that greatly improves the calculation proficiency of logic nanodevices without the confinement of the lithography process in nanotechnology.

Catalyst-based biomolecular logic gates.

Regulatory processes in biology can be re-conceptualized in terms of logic gates, analogous to those in computer science. Frequently, biological systems need to respond to multiple, sometimes conflicting, inputs to provide the correct output. The language of logic gates can then be used to model complex signal transduction and metabolic processes. Advances in synthetic biology in turn can be used to construct new logic gates, which find a variety of biotechnology applications including in the production of high value chemicals, biosensing and drug delivery. In this review, we focus on advances in the construction of logic gates that take advantage of biological catalysts, including both protein-based and nucleic acid-based enzymes. These catalyst-based biomolecular logic gates can read a variety of molecular inputs and provide chemical, optical and electrical outputs, allowing them to interface with other types of biomolecular logic gates or even extend to inorganic systems. Continued advances in molecular modeling and engineering will facilitate the construction of new logic gates, further expanding the utility of biomolecular computing.

DNA Matrix Operation Based on the Mechanism of the DNAzyme Binding to Auxiliary Strands to Cleave the Substrate.

Numerical computation is a focus of DNA computing, and matrix operations are among the most basic and frequently used operations in numerical computation. As an important computing tool, matrix operations are often used to deal with intensive computing tasks. During calculation, the speed and accuracy of matrix operations directly affect the performance of the entire computing system. Therefore, it is important to find a way to perform matrix calculations that can ensure the speed of calculations and improve the accuracy. This paper proposes a DNA matrix operation method based on the mechanism of the DNAzyme binding to auxiliary strands to cleave the substrate. In this mechanism, the DNAzyme binding substrate requires the connection of two auxiliary strands. Without any of the two auxiliary strands, the DNAzyme does not cleave the substrate. Based on this mechanism, the multiplication operation of two matrices is realized; the two types of auxiliary strands are used as elements of the two matrices, to participate in the operation, and then are combined with the DNAzyme to cut the substrate and output the result of the matrix operation. This research provides a new method of matrix operations and provides ideas for more complex computing systems.