Proficiently partitioning of bioactive peptide-ssDNA conjugates by microbead-assisted capillary electrophoresis (MACE).

Low-molecular drug discovery using DNA-encoded chemical library (DEL) is a powerful technology, although improving the partitioning efficiency of affinity ligands from DEL remains a challenge. Here, we assessed the usefulness of microbead-assisted capillary electrophoresis (MACE) for partitioning peptide-oligonucleotide conjugates (POCs), in which high selection pressure is applied because of different mobility of target-modified beads and POCs during CE. Despite their different charge characteristics, all POCs were well separated from the beads.

A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice.

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote.

Steric hindrance and structural flexibility shape the functional properties of a guanine-rich oligonucleotide.

Ligand/protein molecular recognition involves a dynamic process, whereby both partners require a degree of structural plasticity to regulate the binding/unbinding event. Here, we present the characterization of the interaction between a highly dynamic G-rich oligonucleotide, M08s-1, and its target protein, human α-thrombin. M08s-1 is the most active anticoagulant aptamer selected thus far.

Discovery of a Highly Specific Anti-methotrexate (MTX) DNA Aptamer for Antibody-Independent MTX Detection.

High-dose methotrexate (MTX) therapy is used to treat a wide variety of cancers such as leukemia and lymphoma, while the resulting high blood concentration of MTX faces a risk of life-threatening side effects, so it is essential to monitor the concentration carefully. Currently, the MTX concentration is measured using antibody-based kits in a clinical setting; however, the heterogeneity and batch-to-batch variation of antibodies potentially compromise the detection limit. Here, we developed MTX detection systems with chemically synthesizable homogeneous oligonucleotides.

Evaluation of G-quartet-forming deoxyguanines in antiparallel G-quadruplexes using optical spectroscopy and deoxyguanine-to-deoxythymidine scanning.

Due to the dynamic conformations of G-quadruplex structures (G4), determining the guanines that form G4 in a guanine-rich sequence is elusive. Here, we report a method for identifying deoxyguanines (dGs) forming antiparallel G4 by optical spectroscopy. The method, referred to as dG-to-deoxythymidine (dT) scanning, compares the spectra between a wild type and a single nucleobase dG-to-dT mutant at all dG positions.

Reduced cytotoxicity of polyethyleneimine by covalent modification of antioxidant and its application to microalgal transformation.

The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology genetic engineering may prove to be promising for the tailored production of novel metabolites.

Accelerated Discovery of Potent Bioactive anti-TNFα Aptamers by Microbead-Assisted Capillary Electrophoresis (MACE)-SELEX.

Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using in vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells.

Single-Round DNA Aptamer Selection by Combined Use of Capillary Electrophoresis and Next Generation Sequencing: An Aptaomics Approach for Identifying Unique Functional Protein-Binding DNA Aptamers.

In DNA aptamer selection, existing methods do not discriminate aptamer sequences based on their binding affinity and function and the reproducibility of the selection is often poor, even for the selection of well-known aptamers like those that bind the commonly used model protein thrombin. In the present study, a novel single-round selection method (SR-CE selection) was developed by combining capillary electrophoresis (CE) with next generation sequencing. Using SR-CE selection, a successful semi-quantitative and semi-comprehensive aptamer selection for thrombin was demonstrated with high reproducibility for the first time.

Investigations of Chirality Effects on Undifferentiated State of Mesenchymal Stem Cells Using Soft Nanofibrous Oligopeptide Hydrogels.

Soft nanofibrous oligopeptide hydrogels consisting of self-assembled l- and d-form Fmoc-Phe-Phe-Cys networks photo-cross-linked by poly(ethylene glycol)-dimethacrylate, which are referred to as l- and d-gel, respectively, were developed for investigation of chirality effects on the undifferentiated state of mesenchymal stem cells. Encapsulated mesenchymal stem cells in d-gel showed slower growth and less spreading, resulting in higher maintenance of the undifferentiated state, compared to in l-gel. This indicates that d-form peptide materials might be useful as scaffold materials for regenerative medicine using stem cells.

Binding and Structural Properties of DNA Aptamers with VEGF-A-Mimic Activity.

Vascular endothelial growth factors (VEGFs) are hypoxia-inducible secreted proteins to promote angiogenesis, in which VEGF-A is an important molecule that binds and activates VEGF receptor-1 (VEGFR-1) and VEGFR-2. In this study, two DNA aptamers, Apt01 and Apt02, were successfully isolated by alternating consecutive systematic evolution of ligands by exponential enrichment (SELEX) against VEGFR-1 and -2 using deep sequencing analysis in an early selection round. Their binding affinities for VEGFR-2 were lower than that of VEGFR-1, which is similar to that of VEGF-A.

Formation of Spherical Palmelloid Colony with Enhanced Lipid Accumulation by Gel Encapsulation of Chlamydomonas debaryana NIES-2212.

Microalgae such as Chlamydomonas reinhardtii accumulate triacylglycerol (TAG), which is a potential source of biofuels, under stress conditions such as nitrogen deprivation, whereas Chlamydomonas debaryana NIES-2212 has previously been identified and characterized as one of the rare species of Chlamydomonas, which massively accumulates TAG in the stationary phase without external stress. As the high density of the cells in the stationary phase was supposed to act as a trigger for the accumulation of TAG in C. debaryana, in this study, C.

Polyethyleneimine-induced astaxanthin accumulation in the green alga Haematococcus pluvialis by increased oxidative stress.

The unicellular green microalga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin under stress conditions such as nitrogen deficiency. In this study, we discovered an astaxanthin accumulation in H. pluvialis cells by the addition of a synthetic cationic polymer, polyethyleneimine (PEI), into the cell culture.

High Enrichment of Nucleobase-modified Aptamers in Early Selection Rounds by Microbeads-assisted Capillary Electrophoresis SELEX.

Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library.

Rapid Formation of Aggregates with Uniform Numbers of Cells Based on Three-dimensional Dielectrophoresis.

We applied a fabrication method for the formation of island organization of cells based on a three-dimensional (3D) device for negative dielectrophoresis (n-DEP) to produce cell aggregates with uniform numbers of cells rapidly and simply. The intersections formed by rotating the interdigitated array (IDA) with two combs of band electrodes on the upper substrate by 90° relative to the IDA with two combs on the lower substrate were prepared in the device. The AC voltage was applied to a comb on the upper substrate and a comb on the lower substrate, while AC voltage with opposite phase was applied to another comb on the upper substrate and another comb on the lower substrate.

Programmable RNA detection with a fluorescent RNA aptamer using optimized three-way junction formation.

RNAs play essential roles in various cellular processes and can be used as biomarkers. Hence, it is important to detect endogenous RNA for understanding diverse cellular functions and diagnosing diseases. To construct a low-cost and easy-to-use RNA detection probe, a chemically unmodified RNA aptamer that binds to a pro-fluorophore to increase its fluorescence is desirable.

Analysis of Chirality Effects on Stem Cell Fate Using Three-dimensional Fibrous Peptide Hydrogels.

Chirality effects on stem cell fate were investigated in three-dimensional culture using soft fibrous hydrogels consisting of self-assembled - and -form Fmoc-Phe-Phe-Cys networks photo-cross-linked by poly(ethylene glycol) (- and -gel, respectively). Encapsulated human bone marrow-derived mesenchymal stem cells were all alive, spread, and grown in both hydrogels. Interestingly, the cells preferably spread and grew in -gel compared to in -gel under mixed induction, and cell osteogenesis and adipogenesis differentiation in -gel were suppressed compared to in -gel.

Label-Free Rapid Separation and Enrichment of Bone Marrow-Derived Mesenchymal Stem Cells from a Heterogeneous Cell Mixture Using a Dielectrophoresis Device.

Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell resource for stem cell-based therapy, which are generally isolated and enriched by the density-gradient method based on cell size and density after collection of tissue samples. Since this method has limitations with regards to purity and repeatability, development of alternative label-free methods for BMSC separation is desired. In the present study, rapid label-free separation and enrichment of BMSCs from a heterogeneous cell mixture with bone marrow-derived promyelocytes was successfully achieved using a dielectrophoresis (DEP) device comprising saw-shaped electrodes.

Estimation of G-quartet-forming guanines in parallel-type G-quadruplexes by optical spectroscopy measurements of their single-nucleobase substitution sequences.

Since much attention has been paid to in vivo biological functions of G-quadruplexes, structural analyses of G-quadruplexes are essential for understanding their functional mechanisms. Here, we established a simple optical-spectroscopy-based method for the estimation of G-quartet-forming guanines in parallel-type G-quadruplexes using measurements of circular dichroism and the thermal melting temperature.

Noninvasive Fingerprinting-Based Tracking of Replicative Cellular Senescence Using a Colorimetric Polyion Complex Array.

A fingerprint-based sensing approach was used to characterize in vitro cellular senescence. Secretion profiles of cultured human fibroblasts in different senescent stages were transformed into colorimetric enzyme-activity fingerprints by applying cell culture media to a polyion complex array. Analysis of the obtained fingerprints using pattern recognition methods, such as linear discriminant analysis and hierarchical clustering analysis, revealed that the polyion complex array allows the noninvasive tracking of the replicative senescence progress even in those stages where a conventional marker such as senescence-associated β-galactosidase is negative.

Bone-targeting poly(ethylene sodium phosphate).

Poly(ethylene sodium phosphate) (PEP·Na) showed excellent cytocompatibility and in vivo bone affinity. Moreover, PEP·Na did not interact with thrombin, which is a coagulation-related protein. Because immobilization of therapeutic agents and imaging probes on PEP·Na is easily performed, PEP·Na is a promising polymer for bone-targeted therapies.