In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system.

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E.coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E.

Each side chain of cyclosporin A is not essential for high passive permeability across lipid bilayers.

We compared the passive permeability of cyclosporin A (CsA) derivatives with side chain deletions across lipid bilayers. CsA maintained passive permeability after losing any one of the side chains, which suggests that the propensity of the backbone of CsA is an important component for high passive permeability.

Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device.

Label-free quantification of passive membrane permeability of cyclic peptides across lipid bilayers: penetration speed of cyclosporin A across lipid bilayers.

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers.

Supramolecular Mechanosensitive Potassium Channel Formed by Fluorinated Amphiphilic Cyclophane.

Inspired by mechanosensitive potassium channels found in nature, we developed a fluorinated amphiphilic cyclophane composed of fluorinated rigid aromatic units connected via flexible hydrophilic octa(ethylene glycol) chains. Microscopic and emission spectroscopic studies revealed that the cyclophane could be incorporated into the hydrophobic layer of the lipid bilayer membranes and self-assembled to form a supramolecular transmembrane ion channel. Current recording measurements using cyclophane-containing planer lipid bilayer membranes successfully demonstrated an efficient transmembrane ion transport.

A microreactor sealing method using adhesive tape for digital bioassays.

Digital assays using microreactors fabricated on solid substrates are useful for carrying out sensitive assays of infectious diseases and other biological tests. However, sealing of the microchambers using fluid oil is difficult for non-experts, and thus hinders the widespread use of digital microreactor assays. Here, we propose the physical isolation of tiny reactors with adhesive tape (PITAT) using simple, commercially available pressure-sensitive adhesive (PSA) tape as a separator of the microreactors.

Amplification of over 100 kbp DNA from Single Template Molecules in Femtoliter Droplets.

Reconstitution of the DNA amplification system in microcompartments is the primary step toward artificial cell construction through a bottom-up approach. However, amplification of >100 kbp DNA in micrometer-sized reactors has not yet been achieved. Here, implementing a fully reconstituted replisome of in micrometer-sized water-in-oil droplets, we developed the replication cycle reaction (RCR) system.

Multidimensional Digital Bioassay Platform Based on an Air-Sealed Femtoliter Reactor Array Device.

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g.

Synthetic Ion Channel Formed by Multiblock Amphiphile with Anisotropic Dual-Stimuli-Responsiveness.

Transmembrane proteins within biological membranes exhibit varieties of important functions that are vital for many cellular activities, and the development of their synthetic mimetics allows for deep understanding in related biological events. Inspired by the structures and functions of natural ion channels that can respond to multiple stimuli in an anisotropic manner, we developed multiblock amphiphile in this study. When was incorporated into the lipid bilayer membranes, formed a supramolecular ion channel whose ion transport property was controllable by the polarity and amplitude of the applied voltage.

Imidazolinium-based Multiblock Amphiphile as Transmembrane Anion Transporter.

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units.

Single Cell Array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based Cell Classification and Selective Photorelease of Cells.

Single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval.

A synthetic ion channel with anisotropic ligand response.

Biological membranes play pivotal roles in the cellular activities. Transmembrane proteins are the central molecules that conduct membrane-mediated biochemical functions such as signal transduction and substance transportation. Not only the molecular functions but also the supramolecular properties of the transmembrane proteins such as self-assembly, delocalization, orientation and signal response are essential for controlling cellular activities.

Revealing the Metabolic Activity of Persisters in Mycobacteria by Single-Cell DO Raman Imaging Spectroscopy.

The metabolic activity of bacterial cells largely differentiates even within a clonal population. Such metabolic divergence among cells is thought to play an important role for phenotypic adaptation to ever-changing environmental conditions, such as antibiotic persistence. It has long been thought that persisters are in a state called dormancy, in which cells are metabolically inactive and do not grow.

Accurate high-throughput screening based on digital protein synthesis in a massively parallel femtoliter droplet array.

We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary.

Mobile imaging platform for digital influenza virus counting.

Droplet-based digital bioassays enable highly sensitive and quantitative analysis of biomolecules, and are thought to be suitable for point-of-care diagnosis. However, digital bioassays generally require fluorescence microscopy for detection, which is too large for point-of-care testing. Here, we developed a simple smartphone-based mobile imaging platform for digital bioassays.

Regeneration of Giant Protoplasts to Their Original Form.

The spheroplasts and protoplasts of cell wall-deficient (CWD) bacteria are able to revert to their original cellular morphologies through the regeneration of their cell walls. However, whether this is true for giant protoplasts (GPs), which can be as large as 10 μm in diameter, is unknown. GPs can be prepared from various bacteria, including and , and also from fungi, through culture in the presence of inhibitors for cell wall synthesis or mitosis.

Osmolyte-Enhanced Protein Synthesis Activity of a Reconstituted Translation System.

Molecular crowding is receiving great attention in cell-free synthetic biology because molecular crowding is a critical feature of natural cell discrimination from artificial cells. Further, it has significant and generic influences on biomolecular functions. Although there are reports on how the macromolecular crowder reagents affect cell-free systems such as transcription and translation, the second class of molecular crowder reagents with low molecular weight, osmolyte, was much less studied in cell-free systems.

Antibody-free digital influenza virus counting based on neuraminidase activity.

There is large demand for a quantitative method for rapid and ultra-sensitive detection of the influenza virus. Here, we established a digital influenza virus counting (DIViC) method that can detect a single virion without antibody. In the assay, a virion is stochastically entrapped inside a femtoliter reactor array device for the fluorogenic assay of neuraminidase, and incubated for minutes.

Hybrid cell reactor system from Escherichia coli protoplast cells and arrayed lipid bilayer chamber device.

We developed a novel hybrid cell reactor system via functional fusion of single Escherichia coli protoplast cells, that are deficient in cell wall and expose plasma membrane, with arrayed lipid bilayer chambers on a device in order to incorporate the full set of cytosolic and membrane constituents into the artificial chambers. We investigated gene expression activity to represent the viability of the hybrid cell reactors: over 20% of hybrid cells showed gene expression activity from plasmid or mRNA. This suggests that the hybrid cell reactors retained fundamental activity of genetic information transduction.

Mechano-Sensitive Synthetic Ion Channels.

Mechanical stress is a ubiquitous stimulus sensed by membrane proteins, but rarely by synthetic molecules. Inspired by mechano-sensitive ion channels found in cell membranes, tension-responsive transmembrane multiblock amphiphiles were developed. In membranes, a single-transmembrane amphiphile responds to both expanding and contracting tensions to weaken and strengthen the stacking of membrane-spanning units, respectively, and ion transportation is triggered by expanding tension to form a supramolecular channel, while little transportation is observed under a tensionless condition.

Reversible ion transportation switch by a ligand-gated synthetic supramolecular ion channel.

Inspired by the regulation of cellular activities found in the ion channel proteins, here we developed membrane-embedded synthetic chiral receptors 1 and 2 with different terminal structures, where receptor 1 has hydrophobic triisopropylsilyl (TIPS) groups and receptor 2 has hydrophilic hydroxy groups. The receptors have ligand-binding units that interact with cationic amphiphiles such as 2-phenethylamine (PA). Conductance study revealed that the receptors hardly show ion transportation at the ligand-free state.

Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions.

Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.