Oxidation-responsive supramolecular hydrogels based on glucosamine derivatives with an aryl sulfide group.

Supramolecular hydrogels can be obtained via self-assembly of small molecules in aqueous environments. In this study, we describe the development of oxidation-responsive supramolecular hydrogels comprising glucosamine derivatives with an aryl sulfide group. We demonstrate that hydrogen peroxide can induce a gel-sol transition through the oxidation of the sulfide group to the corresponding sulfoxide. Furthermore, we show that this oxidation responsiveness can be extended to photo-responsiveness with the aid of a photosensitizer.

Installing Reduction Responsiveness into Biomolecules by Introducing Nitroaryl Groups.

Artificially synthesized stimuli-responsive biomolecules are attractive as molecular tools for monitoring and modulating biological systems. In biological systems, redox stimuli are common, and their dysregulation is typically linked to various abnormal or disease states. In this Concept article, the molecular design of reduction-responsive biomolecules, such as peptides, nucleic acids, and saccharides, which are produced by introducing nitroaryl groups into them, is reviewed with a special emphasis on simple 4-nitrobenzene-based motifs.

Reliable and sensitive detection of glycosaminoglycan chains with immunoblots.

Complex glycans play vital roles in many biological processes, ranging from intracellular signaling and organ development to tumor growth. Glycan expression is routinely assessed by the application of glycan-specific antibodies to cells and tissues. However, glycan-specific antibodies quite often show a large number of bands on immunoblots and it is hard to interpret the data when reliable controls are lacking. This limits the scope of glycobiology studies and poses challenges for replication. We sought to resolve this issue by developing a novel strategy that utilizes an immunoreaction enhancing technology to vastly improve the speed and quality of glycan-based immunoblots. As a representative case study, we used chondroitin sulfate glycosaminoglycan (CS-GAG) chains as the carbohydrate target and a monoclonal antibody, CS-56, as the probe. We discovered that preincubation of the antibody with its antigenic CS-GAG chain distinguishes true-positive signals from false-positive ones. We successfully applied this strategy to 10E4, a monoclonal anti heparan sulfate GAGs (HS-GAGs) antibody, where true-positive signals were confirmed by chemical HS-GAG depolymerization on the membrane. This evidence that glycan-specific antibodies can generate clear and convincing data on immunoblot with highly replicable results opens new opportunities for many facets of life science research in glycobiology.

Supramolecular Architectures of Nucleic Acid/Peptide Hybrids.

Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.

Hybrid Soft Nanomaterials Composed of DNA Microspheres and Supramolecular Nanostructures of Semi-artificial Glycopeptides.

Aqueous hybrid soft nanomaterials consisting of plural supramolecular architectures with a high degree of segregation (orthogonal coexistence) and precise hierarchy at the nano- and microscales, which are reminiscent of complex biomolecular systems, have attracted increasing attention. Remarkable progress has been witnessed in the construction of DNA nanostructures obtained by rational sequence design and supramolecular nanostructures of peptide derivatives through self-assembly under aqueous conditions. However, orthogonal self-assembly of DNA nanostructures and supramolecular nanostructures of peptide derivatives in a single medium has not yet been explored in detail. In this study, DNA microspheres, which can be obtained from three single-stranded DNAs, and three different supramolecular nanostructures (helical nanofibers, straight nanoribbons, and flowerlike microaggregates) of semi-artificial glycopeptides were simultaneously constructed in a single medium by a simple thermal annealing process, which gives rise to hybrid soft nanomaterials. Fluorescence imaging with selective staining of each supramolecular nanostructure uncovered the orthogonal coexistence of these structures with only marginal impact on their morphology. Additionally, the biostimuli-responsive degradation propensity of each supramolecular architecture is retained, and this may allow the construction of active soft nanomaterials exhibiting intelligent biofunctions.

A disordered tether to iLID improves photoswitchable protein patterning on model membranes.

Reversible protein patterning on model membranes is important to reproduce spatiotemporal protein dynamics in vitro. An engineered version of iLID, disiLID, with a disordered domain as a membrane tether improves the recruitment of Nano under blue light and the reversibility in the dark, which enables protein patterning on membranes with higher spatiotemporal precision.

Design of supramolecular hybrid nanomaterials comprising peptide-based supramolecular nanofibers and in situ generated DNA nanoflowers through rolling circle amplification.

The artificial construction of multicomponent supramolecular materials comprising plural supramolecular architectures that are assembled orthogonally from their constituent molecules has attracted growing attention. Here, we describe the design and development of multicomponent supramolecular materials by combining peptide-based self-assembled fibrous nanostructures with globular DNA nanoflowers constructed by the rolling circle amplification reaction. The orthogonally constructed architectures were dissected by fluorescence imaging using the selective fluorescence staining procedures adapted to this study. The present, unique hybrid materials developed by taking advantage of each supramolecular architecture based on their peptide and DNA functions may offer distinct opportunities to explore their bioapplications as a soft matrix.

Enhancing Electrochemical Biosensor Performance for 17ß-Estradiol Determination with Short Split-Aptamers.

Chronic exposure of 17ß-estradiol (E2) even at low concentration can disorganize the endocrine system and lead to undesirable health problems in the long run. An electrochemical biosensor for rapid detection of E2 in water samples was successfully developed. The biosensor was based on split DNA aptamers attached onto poly (methacrylic acid-co-n butyl acrylate-succinimide) microspheres deposited on polypyrrole nanowires coated electrode (PPY/PMAA-NBA). The sandwich paired of split DNA aptamers used were truncated from 75 mer parent aptamers. These two strands of 12-mer and 14-mer split DNA aptamers were then immobilized on the PMAA-NBA microspheres. In the presence of E2, the split DNA aptamers formed an apt12-E2-apt14 complex, where the binding reaction on the electrode surface led to the detection of E2 by differential pulse voltammetry using ferrocyanide as a redox indicator. Under optimum conditions, the aptasensor detected E2 concentrations in the range of 1 × 10-4 M to 1 × 10-12 M (R2 = 0.9772) with a detection limit of 4.8 × 10-13 M. E2, which were successfully measured in a real sample with 97-104% recovery and showed a good correlation (R2 = 0.9999) with the established method, such as high-performance liquid chromatography. Interactions between short and sandwich-type aptamers (split aptamers) demonstrated improvement in aptasensor performance, especially the selectivity towards several potential interferents.

Construction of a Reduction-responsive DNA Microsphere using a Reduction-cleavable Spacer based on a Nitrobenzene Scaffold.

Here, we describe the design and synthesis of a new reduction-cleavable spacer (RCS) based on a nitrobenzene scaffold for constructing reduction-responsive oligonucleotides according to standard phosphoramidite chemistry. In addition, we demonstrate that the introduction of the RCS in the middle of an oligonucleotide (30 nt) enables the construction of a self-assembled microsphere capable of exhibiting a reduction-responsive disassembly.

Development of an Amino Sugar-Based Supramolecular Hydrogelator with Reduction Responsiveness.

Stimuli-responsive supramolecular hydrogels are a newly emerging class of aqueous soft materials with a wide variety of bioapplications. Here we report a reduction-responsive supramolecular hydrogel constructed from a markedly simple low-molecular-weight hydrogelator, which is developed on the basis of modular molecular design containing a hydrophilic amino sugar and a reduction-responsive nitrophenyl group. The hydrogel formation ability differs significantly between glucosamine- and galactosamine-based self-assembling molecules, which are epimers at the C4 position, and only the glucosamine-based derivative can act as a hydrogelator.

One-Pot Construction of Multicomponent Supramolecular Materials Comprising Self-Sorted Supramolecular Architectures of DNA and Semi-Artificial Glycopeptides.

Multicomponent supramolecular materials, which comprise plural supramolecular architectures that exhibit distinct self-sorting behaviors, are receiving increasing attention because they can be implemented with sophisticated functions and hierarchical structures, e.g., living cells. Nevertheless, the application of supramolecular system design to engineer self-sorting behaviors among plural supramolecular architectures remains challenging. Herein, we show that the thermal annealing-induced one-pot assembly of multiple single-stranded DNAs and a single semi-artificial glycopeptide (GP) results in the emergent formation of integrative self-sorted supramolecular nanostructures (ssSNs) consisting of a GP supramolecular nanoribbon surrounded by DNA tile-nanotubes. Fluorescence imaging revealed the formation of each supramolecular nanostructure through orthogonal molecular assembling processes. Moreover, the fluorescence recovery after photobleaching (FRAP) disclosed the presence of reversible attractive interactions between the DNA tile, prior to the formation of the tile-nanotube, and the GP supramolecular nanostructures at the mesoscale level, which are crucial for the formation of the integrative ssSNs. Further, we revealed that the integrative ssSNs retain their biostimuli responsiveness so that each supramolecular nanostructure can be selectively degraded. Finally, we successfully constructed a complex soft nanomaterial composed of ternary supramolecular architectures (a GP supramolecular nanostructure, DNA tile-nanotube, and DNA microsphere) based on the present as well as previous findings.

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

Old but not obsolete: an enhanced high-speed immunoblot.

The immunoblotting technique (also known as western blotting) is an essential tool used in biomedical research to determine the relative size and abundance of specific proteins and protein modifications. However, long incubation times severely limit its throughput. We have devised a system that improves antigen binding by cyclic draining and replenishing (CDR) of the antibody solution in conjunction with an immunoreaction enhancing agent. Biochemical analyses revealed that the CDR method reduced the incubation time of the antibodies, and the presence of a commercial immunoreaction enhancing agent altered the affinity of the antibody, respectively. Combination of the CDR method with the immunoreaction enhancing agent considerably enhanced the output signal and further reduced the incubation time of the antibodies. The resulting high-speed immunoblot can be completed in 20 min without any loss in sensitivity. Further, the antibodies are fully reusable. This method is effective for both chemiluminescence and fluorescence detection. Widespread adoption of this technique could dramatically boost efficiency and productivity across the life sciences.