A bioinspired bifunctional catalyst: an amphiphilic organometallic catalyst for ring-closing metathesis forming liquid droplets in aqueous media.

Inspired by phase-separated biopolymers with enzymatic activity, we developed an amphiphilic catalyst consisting of alternating hydrophilic oligo(ethylene glycol) and hydrophobic aromatic units bearing a Hoveyda-Grubbs catalyst center (MAHGII). MAHGII served as both a droplet-forming scaffold and a catalyst for ring-closing metathesis reactions, providing a new biomimetic system that promotes organic reactions in an aqueous environment.

Transient formation of multi-phase droplets caused by the addition of a folded protein into complex coacervates with an oppositely charged surface relative to the protein.

Complex coacervates have received increasing attention due to their use as simple models of membrane-less organelles and microcapsule platforms. The incorporation of proteins into complex coacervates is recognized as a crucial event that enables understanding of membrane-less organelles in cells and controlling microcapsules. Here, we investigated the incorporation of proteins into complex coacervates with a focus on the progress of the incorporation process. This stands in contrast to most previous studies, which have been focused the endpoint of the incorporation process. For that purpose, client proteins, i.e., lysozyme, ovalbumin, and pyruvate oxidase, were mixed with complex coacervate scaffolds consisting of two polyelectrolytes, i.e., the positively charged poly(diallyldimethylammonium chloride) and the negatively charged carboxymethyl dextran sodium salt, and the process was studied. Spectroscopic analysis and microscopic imaging demonstrated that electrostatic factors are the primary driving force of the incorporation of the client proteins into the complex coacervate scaffolds. Moreover, we discovered the formation of multi-phase droplets when a charged protein was incorporated into a complex coacervate whose surface was charged oppositely relative to that of the protein. The droplets inside the complex coacervates were found to be the diluted phase trapped as internal vacuoles. These findings provide fundamental insight into the temporal changes at the droplet interface during the incorporation of proteins into complex coacervates. This knowledge will facilitate the understanding of biological events associated with membrane-less organelles and will contribute to the industrial development of the use of microcapsules.

Dense and Acidic Organelle-Targeted Visualization in Living Cells: Application of Viscosity-Responsive Fluorescence Utilizing Restricted Access to Minimum Energy Conical Intersection.

Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized AnP2-H and AnP2-OEG, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(N,N-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection. AnP2-H and AnP2-OEG exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems, AnP2-OEG showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.

Phase-Separation Propensity of Non-ionic Amino Acids in Peptide-Based Complex Coacervation Systems.

Uncovering the sequence-encoded molecular grammar that governs the liquid-liquid phase separation (LLPS) of proteins is a crucial issue to understand dynamic compartmentalization in living cells and the emergence of protocells. Here, we present a model LLPS system that is induced by electrostatic interactions between anionic nucleic acids and cationic oligolysine peptides modified with 12 different non-ionic amino acids, with the aim of creating an index of "phase-separation propensity" that represents the contribution of non-ionic amino acids to LLPS. Based on turbidimetric titrations and microscopic observations, the lower critical peptide concentrations where LLPS occurs (Ccrit) were determined for each peptide. A correlation analysis between these values and known amino acid indices unexpectedly showed that eight non-ionic amino acids inhibit the generation of LLPS, whereby the extent of inhibition increases with increasing hydrophobicity of the amino acids. However, three aromatic amino acids deviate from this trend and rather markedly promote LLPS despite their high hydrophobicity. A comparison with double-stranded DNA and polyacrylic acid revealed that this is primarily due to interactions with DNA nucleobases. Our approach to quantify the contribution of non-ionic amino acids can be expected to help to provide a more accurate description and prediction of the LLPS propensity of peptides/proteins.

Damage-free evaluation of cultured cells based on multivariate analysis with a single-polymer probe.

We present a pattern-recognition-based sensor that targets cell-derived components in culture media and evaluates cultured cells without damaging them. An array sensor made of a single-polymer probe was employed to obtain fluorescence response patterns of the analyte media, allowing successful identification of the type and state of the cells via multivariate analysis.

Uncharged Components of Single-Stranded DNA Modulate Liquid-Liquid Phase Separation With Cationic Linker Histone H1.

Liquid-liquid phase separation (LLPS) of proteins and DNAs has been recognized as a fundamental mechanism for the formation of intracellular biomolecular condensates. Here, we show the role of the constituent DNA components, i.e., the phosphate groups, deoxyribose sugars, and nucleobases, in LLPS with a polycationic peptide, linker histone H1, a known key regulator of chromatin condensation. A comparison of the phase behavior of mixtures of H1 and single-stranded DNA-based oligomers in which one or more of the constituent moieties of DNA were removed demonstrated that not only the electrostatic interactions between the anionic phosphate groups of the oligomers and the cationic residues of H1, but also the interactions involving nucleobases and deoxyriboses (i) promoted the generation of spherical liquid droplets via LLPS as well as (ii) increased the density of DNA and decreased its fluidity within the droplets under low-salt conditions. Furthermore, we found the formation of non-spherical assemblies with both mobile and immobile fractions at relatively higher concentrations of H1 for all the oligomers. The roles of the DNA components that promote phase separation and modulate droplet characteristics revealed in this study will facilitate our understanding of the formation processes of the various biomolecular condensates containing nucleic acids, such as chromatin organization.

Microfluidic Sensing System with a Multichannel Surface Plasmon Resonance Chip: Damage-Free Characterization of Cells by Pattern Recognition.

The development of a versatile sensing strategy for the damage-free characterization of cultured cells is of great importance for both fundamental biological research and industrial applications. Here, we present a pattern-recognition-based cell-sensing approach using a multichannel surface plasmon resonance (SPR) chip. The chip, in which five cysteine derivatives with different structures are immobilized on Au films, is capable of generating five unique SPR sensorgrams for the cell-secreted molecules that are contained in cell culture media. An automatic statistical program was built to acquire kinetic parameters from the SPR sensorgrams and to select optimal parameters as "pattern information" for subsequent multivariate analysis. Our system rapidly (∼10 min) provides the complex information by merely depositing a small amount of cell culture media (∼25 µL) onto the chip, and the amount of information obtained is comparable to that furnished by a combination of conventional laborious biochemical assays. This noninvasive pattern-recognition-based cell-sensing approach could potentially be employed as a versatile tool for characterizing cells.

A Multichannel Pattern-Recognition-Based Protein Sensor with a Fluorophore-Conjugated Single-Stranded DNA Set.

Recently, pattern-recognition-based protein sensing has received considerable attention because it offers unique opportunities that complement more conventional antibody-based detection methods. Here, we report a multichannel pattern-recognition-based sensor using a set of fluorophore-conjugated single-stranded DNAs (ssDNAs), which can detect various proteins. Three different fluorophore-conjugated ssDNAs were placed into a single microplate well together with a target protein, and the generated optical response pattern that corresponds to each environment-sensitive fluorophore was read via multiple detection channels. Multivariate analysis of the resulting optical response patterns allowed an accurate detection of eight different proteases, indicating that fluorescence signal acquisition from a single compartment containing a mixture of ssDNAs is an effective strategy for the characterization of the target proteins. Additionally, the sensor could identify proteins, which are potential targets for disease diagnosis, in a protease and inhibitor mixture of different composition ratios. As our sensor benefits from simple construction and measurement procedures, and uses accessible materials, it offers a rapid and simple platform for the detection of proteins.

Pattern-recognition-based Sensor Arrays for Cell Characterization: From Materials and Data Analyses to Biomedical Applications.

To capture a broader scope of complex biological phenomena, alternatives to conventional sensing based on specificity for cell detection and characterization are needed. Pattern-recognition-based sensing is an analytical method designed to mimic mammalian sensory systems for analyte identification based on the pattern recognition of multivariate data, which are generated using an array of multiple probes that cross-reactively interact with analytes. This sensing approach is significantly different from conventional specific cell sensing based on highly specific probes, including antibodies against biomarkers. Encouraged by the advantages of this technique, such as the simplicity, rapidity, and tunability of the systems without requiring a priori knowledge of biomarkers, numerous sensor arrays have been developed over the past decade and used in a variety of cell sensing applications; these include disease diagnosis, drug discovery, and fundamental research. This review summarizes recent progress in pattern-recognition-based cell sensing, with a particular focus on guidelines for designing materials and arrays, techniques for analyzing response patterns, and applications of sensor systems that are focused primarily for the biomedical field.

Optical Fingerprints of Proteases and Their Inhibited Complexes Provided by Differential Cross-Reactivity of Fluorophore-Labeled Single-Stranded DNA.

The detection of proteases and their complexes with inhibitor proteins is of great importance for diagnosis and medical-treatment applications. In this study, we report a fingerprint-based sensor using an array of single-stranded DNAs (ssDNAs) labeled with environment-responsive 3'-carboxytetramethylrhodamine (TAMRA) for the identification of proteases. Four TAMRA-modified ssDNAs with different sequences solubilized in two different buffer solutions were incorporated in an array that was capable of generating fluorescent fingerprints unique to the proteases through diverse cross-reactive interactions, allowing the discrimination of (i) 8 proteases and (ii) 12 different mixtures of trypsin and its inhibitor protein (α1-antitrypsin) by multivariate analysis. Constructing an array with TAMRA-modified DNA aptamers that bind to different sites of human thrombin provides fluorescence fingerprints that reflect a reduction of the exposed surface area of thrombin upon complexation with antithrombin III, even in the presence of human serum. We finally demonstrate the potential of hybridization with complementary DNAs as an effective means to easily double the fingerprint information for proteases. Our approach based on the cross-reactive capability of ssDNAs enables high-throughput fingerprint-based sensing that can be flexibly designed and easily constructed, not only for the identification of a variety of proteins including proteases but also for the evaluation of their complexation ability.

One-Component Array Based on a Dansyl-Modified Polylysine: Generation of Differential Fluorescent Signatures for the Discrimination of Human Cells.

A one-component array-based sensor consisting of a dansyl-modified polylysine (PLL-Dnc) is capable of discriminating the types and compositional ratios of human cells using varying buffer conditions. The PLL-Dnc sensor array, which affords turn-on fluorescence responses against analyte cells that depend on the pH value and the ionic strength, generates differential fluorescence signatures of cells and successfully discriminates eight types of human cell lines (2.0 × 104 cells/mL) with 100% accuracy using multivariate analysis. The array also allows differentiation of the composition of the cell mixtures that contain cells with the same tissue origin but of different subtypes. The good discrimination ability and simple platform of our "one-component"-based array allows an easy and rapid sensing of cells without requiring any information on specific biomarkers.