Histamine-Responsive Hydrogel Biosensors Based on Aptamer Recognition and DNA-Driven Swelling Hydrogels.

Detection of chemical substances is essential for living a healthy and cultural life in the modern world. One type of chemical sensing technology, biosensing, uses biological components with molecular recognition abilities, enabling a broad spectrum of sensing targets. Short single-stranded nucleic acids called aptamers are one of the biological molecules used in biosensing, and sensing methods combining aptamers and hydrogels have been researched for simple sensing applications. In this research, we propose a hydrogel-based biosensor that uses aptamer recognition and DNA-driven swelling hydrogels for the rapid detection of histamine. Aptamer recognition and DNA-driven swelling hydrogels are directly linked via DNA molecular reactions, enabling rapid sensing. We selected histamine, a major food poisoning toxin, as our sensing target and detected the existence of histamine within 10 min with significance. Because this sensing foundation uses aptamers, which have a vast library of targets, we believe this system can be expanded to various targets, broadening the application of hydrogel-based biosensors.

Programmable Computational RNA Droplets Assembled via Kissing-Loop Interaction.

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.

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.

Scanning electrochemical microscopy for determining oxygen consumption rates of cells in hydrogel fibers fabricated using an extrusion 3D bioprinter.

Three-dimensional (3D)-cultured cells have attracted the attention of researchers in tissue engineering- and drug screening-related fields. Among them, 3D cellular fibers have attracted significant attention because they can be stacked to prepare more complex tissues and organs. Cellular fibers are widely fabricated using extrusion 3D bioprinters. For these applications, it is necessary to evaluate cellular activities, such as the oxygen consumption rate (OCR), which is one of the major metabolic activities. We previously reported the use of scanning electrochemical microscopy (SECM) to evaluate the OCRs of cell spheroids. However, the SECM approach has not yet been applied to hydrogel fibers prepared using the bioprinters. To the best of our knowledge, this is the first study to evaluate the OCR of cellular fibers printed by extrusion 3D bioprinters. First, the diffusion theory was discussed to address this issue. Next, diffusion models were simulated to compare realistic models with this theory. Finally, the OCRs of MCF-7 cells in the printed hydrogel fibers were evaluated as a proof of concept. Our proposed approach could potentially be used to evaluate the OCRs of tissue-engineered fibers for organ transplantation and drug screening using in-vitro models.

Transient control of lytic activity via a non-equilibrium chemical reaction system.

The development of artificial non-equilibrium chemical reaction systems has recently attracted considerable attention as a new type of biomimetic. However, due to the lack of bioorthogonality, such reaction systems could not be linked to the regulation of any biological phenomena. Here, we have newly designed a non-equilibrium reaction system based on olefin metathesis to produce the Triton X-mimetic non-ionic amphiphile as a kinetic product. Using phospholipid vesicles encapsulating fluorescent dyes and red blood cells as cell models, we demonstrate that the developed chemical reaction system is applicable for transient control of the resulting lytic activity.

DNA droplets for intelligent and dynamical artificial cells: from the viewpoint of computation and non-equilibrium systems.

Living systems are molecular assemblies whose dynamics are maintained by non-equilibrium chemical reactions. To date, artificial cells have been studied from such physical and chemical viewpoints. This review briefly gives a perspective on using DNA droplets in constructing artificial cells. A DNA droplet is a coacervate composed of DNA nanostructures, a novel category of synthetic DNA self-assembled systems. The DNA droplets have programmability in physical properties based on DNA base sequence design. The aspect of DNA as an information molecule allows physical and chemical control of nanostructure formation, molecular assembly and molecular reactions through the design of DNA base pairing. As a result, the construction of artificial cells equipped with non-equilibrium behaviours such as dynamical motions, phase separations, molecular sensing and computation using chemical energy is becoming possible. This review mainly focuses on such dynamical DNA droplets for artificial cell research in terms of computation and non-equilibrium chemical reactions.

Fabrication and Cell Culture Applications of Core-Shell Hydrogel Fibers Composed of Chitosan/DNA Interfacial Polyelectrolyte Complexation and Calcium Alginate: Straight and Beaded Core Variations.

Core-shell hydrogel fibers are widely used in cell culture applications. A simple and rapid method is presented for fabricating core-shell hydrogel fibers, consisting of straight or beaded core fibers, for cell culture applications. The core fibers are prepared using interfacial polyelectrolyte complexation (IPC) with chitosan and DNA. Briefly, two droplets of chitosan and DNA are brought in contact to form an IPC film, which is dragged to prepare an IPC fiber. The incubation time and DNA concentration are adjusted to prepare straight and beaded IPC fibers. The fibers with Ca2+ are immersed in an alginate solution to form calcium alginate shell hydrogels around the core IPC fibers. To the best of the knowledge, this is the first report of core-shell hydrogel fibers with IPC fiber cores. To demonstrate cell culture, straight hydrogel fibers are applied to fabricate hepatic models consisting of HepG2 and 3T3 fibroblasts, and vascular models consisting of human umbilical vein endothelial cells and 3T3 fibroblasts. To evaluate the effect of co-culture, albumin secretion, and angiogenesis are evaluated. Beaded hydrogel fibers are used to fabricate many size-controlled spheroids for fiber and cloning applications. This method can be widely applied in tissue engineering and cell analysis.

Control of Reversible Formation and Dispersion of the Three Enzyme Networks Integrating DNA Computing.

The majority of biological reactions in the cytoplasm of living cells occur via enzymatic cascade reactions. To achieve efficient enzyme cascade reactions mimicking the proximity conditions of enzymes in the cytoplasm, the proximity of each enzyme, creating a high local concentration of proteins, has been recently investigated using the conjugation of synthetic polymer molecules, proteins, and nucleic acids. Although there have been methodologies reported for the complex formation and enhanced activity of cascade reactions due to the proximity of each enzyme using DNA nanotechnology, one pair of the enzyme (GOx and HRP) complex is only assembled by the mutual independence of various shapes of the DNA structure. This study reports the network formation of three enzyme complexes assembled by a triple-branched DNA structure as a unit, thus enabling the reversible formation and dispersion of the three enzyme complex networks using single-stranded DNA, RNA, and enzymes. It was found that the activities of the three enzyme cascade reactions in the enzyme-DNA complex network were controlled by formation and dispersion of the three enzyme complex networks, due to the proximity of each enzyme with the enzyme-DNA complex network. Furthermore, three micro RNA sequences for breast cancer biomarkers were successfully detected using an enzyme-DNA complex network integrated with DNA computing. Overall, the reversible formation and dispersion of the enzyme-DNA complex network through the external stimulation of biomolecules and DNA computing provide a novel platform for controlling the production amount, diagnosis, theranostics, and biological or environmental sensing.

Sequence-dependent fusion dynamics and physical properties of DNA droplets.

Liquid-liquid phase separation (LLPS) of biopolymer molecules generates liquid-like droplets. Physical properties such as viscosity and surface tension play important roles in the functions of these droplets. DNA-nanostructure-based LLPS systems provide useful model tools to investigate the influence of molecular design on the physical properties of the droplets, which has so far remained unclear. Herein, we report changes in the physical properties of DNA droplets by sticky end (SE) design in DNA nanostructures. We used a Y-shaped DNA nanostructure (Y-motif) with three SEs as a model structure. Seven different SE designs were used. The experiments were performed at the phase transition temperature where the Y-motifs self-assembled into droplets. We found that the DNA droplets assembled from the Y-motifs with longer SEs exhibited a longer coalescence period. In addition, the Y-motifs with the same length but different sequence SEs showed slight variations in the coalescence period. Our results suggest that the SE length greatly affected the surface tension at the phase transition temperature. We believe that these findings will accelerate our understanding of the relationship between molecular design and the physical properties of droplets formed via LLPS.

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.

Water-in-Water Droplets Selectively Uptake Self-Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology.

DNA is an excellent material for constructing self-assembled nano/microstructures. Owing to the widespread use of DNA as a building block in laboratories and industry, it is desirable to increase the efficiency of all steps involved in producing self-assembled DNA structures. One of the bottlenecks is the purification required to separate the excess components from the target structures. This paper describes a purification method based on the fractionation by water-in-water (W/W) droplets composed of phase-separated dextran-rich droplets in a polyethylene glycol (PEG)-rich continuous phase. The dextran-rich droplets facilitate the selective uptake of self-assembled DNA nano/microstructures and allow the separation of the target structure. This study investigates the ability to purify DNA origami, DNA hydrogels, and DNA microtubes. The W/W-droplet fractionation allows the purification of structures of a broad size spectrum without changes to the protocol. By quantifying the activity of deoxyribozyme-modified DNA origami after W/W-droplet purification, this study demonstrates that this method sufficiently preserves the accessibility to the surface of a functional DNA nanostructure. It is considered that the W/W-droplet fractionation could become one of the standard methods for the purification of self-assembled DNA nano/microstructures for biomedical and nanotechnology applications owing to its low cost and simplicity.

Repeatable detection of Ag+ ions using a DNA aptamer-linked hydrogel biochemical sensor integrated with microfluidic heating system.

This paper describes repeatable detection of Ag+ ions using a DNA aptamer-linked hydrogel biochemical sensor integrated with a microfluidic heating system. Biochemical sensors that respond to chemical compounds and produce detectable signals have a critical role in many aspects of modern society. In particular, the repeatable measurement of environmental information such as toxic substances including Ag+ ions could be expected to improve the environment. The DNA aptamer is an attractive candidate because of the stability and the selectivity of binding to chemicals. However, previous DNA aptamer biochemical sensors could not measure repeatedly because those sensors did not have initializing functions. To overcome this challenge, we proposed a DNA aptamer-linked hydrogel biochemical sensor integrated with the microfluidic heating system enabling repeatable detection of Ag+ ions. The binding Ag+ ions are dissociated by heating and flushing through the integrated microfluidic heating device. The DNA aptamer-linked hydrogel had the capability to detect a wide range of Ag+ ion concentrations (10-5-10 mM) including a toxic range for various aquatic organisms. Finally, we demonstrated the repeatable detection of the Ag+ ions. These results indicated that our proposed biochemical sensor is expected to use for long-term monitoring with high stability in ambient temperature and low power consumption.

Electrochemical Glue for Binding Chitosan-Alginate Hydrogel Fibers for Cell Culture.

Three-dimensional organs and tissues can be constructed using hydrogels as support matrices for cells. For the assembly of these gels, chemical and physical reactions that induce gluing should be induced locally in target areas without causing cell damage. Herein, we present a novel electrochemical strategy for gluing hydrogel fibers. In this strategy, a microelectrode electrochemically generated HClO or Ca2+, and these chemicals were used to crosslink chitosan-alginate fibers fabricated using interfacial polyelectrolyte complexation. Further, human umbilical vein endothelial cells were incorporated into the fibers, and two such fibers were glued together to construct "+"-shaped hydrogels. After gluing, the hydrogels were embedded in Matrigel and cultured for several days. The cells spread and proliferated along the fibers, indicating that the electrochemical glue was not toxic toward the cells. This is the first report on the use of electrochemical glue for the assembly of hydrogel pieces containing cells. Based on our results, the electrochemical gluing method has promising applications in tissue engineering and the development of organs on a chip.

Capsule-like DNA Hydrogels with Patterns Formed by Lateral Phase Separation of DNA Nanostructures.

Phase separation is a key phenomenon in artificial cell construction. Recent studies have shown that the liquid-liquid phase separation of designed-DNA nanostructures induces the formation of liquid-like condensates that eventually become hydrogels by lowering the solution temperature. As a compartmental capsule is an essential artificial cell structure, many studies have focused on the lateral phase separation of artificial lipid vesicles. However, controlling phase separation using a molecular design approach remains challenging. Here, we present the lateral liquid-liquid phase separation of DNA nanostructures that leads to the formation of phase-separated capsule-like hydrogels. We designed three types of DNA nanostructures (two orthogonal and a linker nanostructure) that were adsorbed onto an interface of water-in-oil (W/O) droplets via electrostatic interactions. The phase separation of DNA nanostructures led to the formation of hydrogels with bicontinuous, patch, and mix patterns, due to the immiscibility of liquid-like DNA during the self-assembly process. The frequency of appearance of these patterns was altered by designing DNA sequences and altering the mixing ratio of the nanostructures. We constructed a phase diagram for the capsule-like DNA hydrogels by investigating pattern formation under various conditions. The phase-separated DNA hydrogels did not only form on the W/O droplet interface but also on the inner leaflet of lipid vesicles. Notably, the capsule-like hydrogels were extracted into an aqueous solution, maintaining the patterns formed by the lateral phase separation. In addition, the extracted hydrogels were successfully combined with enzymatic reactions, which induced their degradation. Our results provide a method for the design and control of phase-separated hydrogel capsules using sequence-designed DNAs. We envision that by incorporating various DNA nanodevices into DNA hydrogel capsules, the capsules will gain molecular sensing, chemical-information processing, and mechanochemical actuating functions, allowing the construction of functional molecular systems.

Aqueous Triple-Phase System in Microwell Array for Generating Uniform-Sized DNA Hydrogel Particles.

DNA hydrogels are notable for their biocompatibility and ability to incorporate DNA information and computing properties into self-assembled micrometric structures. These hydrogels are assembled by the thermal gelation of DNA motifs, a process which requires a high salt concentration and yields polydisperse hydrogel particles, thereby limiting their application and physicochemical characterization. In this study, we demonstrate that single, uniform DNA hydrogel particles can form inside aqueous/aqueous two-phase systems (ATPSs) assembled in a microwell array. In this process, uniform dextran droplets are formed in a microwell array inside a microfluidic device. The dextran droplets, which contain DNA motifs, are isolated from each other by an immiscible PEG solution containing magnesium ions and spermine, which enables the DNA hydrogel to undergo gelation. Upon thermal annealing of the device, we observed the formation of an aqueous triple-phase system in which uniform DNA hydrogel particles (the innermost aqueous phase) resided at the interface of the aqueous two-phase system of dextran and PEG. We expect ATPS microdroplet arrays to be used to manufacture other hydrogel microparticles and DNA/dextran/PEG aqueous triple-phase systems to serve as a highly parallel model for artificial cells and membraneless organelles.

System concentration shift as a regulator of transcription-translation system within liposomes.

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

Editorial on the Special Issue on Recent Advances of Molecular Machines and Molecular Robots.

Molecular machines and molecular robots are a highly interdisciplinary research field including material science, chemistry, biotechnology, biophysics, soft matter physics, micro-electromechanical systems (MEMS), and computer science [...].

Quantification of the effect of site-specific histone acetylation on chromatin transcription rate.

Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ∼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.

Sequence-based engineering of dynamic functions of micrometer-sized DNA droplets.

DNA has the potential to achieve a controllable macromolecular structure, such as hydrogels or droplets formed through liquid-liquid phase separation (LLPS), as the design of its base sequence can result in programmable interactions. Here, we constructed "DNA droplets" via LLPS of sequence-designed DNA nanostructures and controlled their dynamic functions by designing their sequences. Specifically, we were able to adjust the temperature required for the formation of DNA droplets by designing the sequences. In addition, the fusion, fission, and formation of Janus-shaped droplets were controlled by sequence design and enzymatic reactions. Furthermore, modifications of proteins with sequence-designed DNAs allowed for their capture into specific droplets. Overall, our results provide a platform for designing and controlling macromolecular droplets via the information encoded in component molecules and pave the way for various applications of sequence-designed DNA such as cell mimics, synthetic membraneless organelles, and artificial molecular systems.