Publications by authors named "Naoto Nemoto"

Although Green Fluorescent Protein (GFP) is useful and most widely used, steric hindrance due to its size and the time required for chromophore formation are complications. However, it is difficult to form chromophores with peptides to reduce the molecular weight. Therefore, we focused on peptides that can become fluorescent by binding to dyes.

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As one of methods for in vitro selection, a flow reactor type washing/selection system seems to be effective, where a ligand library is composed of "genotype-phenotype linking molecules". In this system, high affinity ligands are selected by their respective "residual ratio" given by exp(-k×t), where k is the dissociation rate constant and t is the washing time. In this paper, we mathematically considered the following possibility.

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Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences.

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Background: Dentinogenic ghost cell tumor is a rare benign tumor that accounts for less than 3% of all cases and consists of the stellate reticulum, which is made up of enamel epithelioid and basaloid cells. Although DGCT is a benign tumor, the local infiltration of the odontogenic epithelium or recurrences have been reported, and its detailed pathology and treatments remain unclear.

Case Presentation: This report describes the case of a 60-year-old Japanese male diagnosed with a maxillary dentinogenic ghost cell tumor.

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Fibroblast growth factor (FGF) is a multifunctional protein that exhibits a wide range of biological effects. Most commonly, it acts as a mitogen, but it also has regulatory, morphological, and endocrine effects. The four receptor subtypes of FGF are activated by more than 20 different FGF ligands.

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cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (10) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3.

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A variable domain of heavy chain antibody (VHH) has different binding properties than conventional antibodies. Conventional antibodies prefer binding to the convex portion of the antigen, whereas VHHs prefer epitopes, such as crevices and clefts on the antigen. Therefore, developing candidates with the binding characteristics of camelid VHHs is important.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction.

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Peptide-based drugs are an attractive new modality of therapeutics, and selection from a large-scale library is a powerful way to identify new lead sequences. In conventional screenings, peptide specificity and stability in physiological heterogenous environments are not evaluated, which sometimes makes subsequent optimization difficult. Here we show that selection using a cDNA display system can be performed in a high percentage of serum and that this might be an option to select molecules with high potency and stability in a biological context.

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Immuno-PCR (IPCR) is a sensitive antigen detection by means of specific antibody-DNA conjugates. To ensure the successful conjugation of a protein (an antibody) with a reporter DNA, immuno-PCR method based on cDNA display (cD-IPCR) has been introduced. The cDNA display molecule is a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level, which is directly used for antigen detection and subsequent qPCR.

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Binding peptides for given target molecules are often selected in vitro during drug discovery and chemical biology research. Among several display technologies for this purpose, complementary DNA (cDNA) display (a covalent complex of a peptide and its encoding cDNA linked via a specially designed puromycin-conjugated DNA) is unique in terms of library size, chemical stability, and flexibility of modification. However, selection of cDNA display libraries often suffers from false positives derived from non-specific binding.

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Efficient and precise construction of DNA libraries is a fundamental starting point for directed evolution of polypeptides. Recently, several in vitro selection methods have been reported that do not rely on cells for protein expression, where peptide libraries in the order of 10 species are used for in vitro affinity selection. To maximize their potential, simple yet versatile construction of DNA libraries from several fragments containing random regions without bacterial transformation is essential.

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Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level.

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Gluten intolerance, or adverse intestinal reactions to gluten, is a fairly common problem among certain groups of people. Celiac disease is the most severe form of gluten intolerance, which can lead to permanent damage in the digestive system. Since lifelong avoidance of gluten is the only available treatment, development of reliable techniques to identify gluten contamination in food is important.

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A cDNA display method was developed based on the mRNA display method to increase its stability and efficiency for the directed evolution of various kinds of peptides and proteins. In this method, the puromycin-linker is a key molecule to realize smart genotype-phenotype coupling. A recently improved puromycin-linker and its use were explained in detail for the in vitro selection of peptides and proteins using the cDNA display method.

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Peptides that recognize artificial materials including synthetic polymers and small molecules are drawing attention in the fields of biotechnology and chemical biology. In particular, reversible peptide aptamers that associate with the target molecules only under specific conditions are interesting. In this work, peptide aptamers that recognize a phenolphthalein derivative (PhP: a pH-sensitive organic dye) immobilized on a solid surface in a pH-dependent manner were selected using an in vitro display method (cDNA display).

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Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level.

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Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents.

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Single-domain antibodies (variable domain of the heavy chain of a heavy chain antibody; VHH) are promising reagents for therapeutics and diagnostics because of their stability, cost-effective production and material workability as a small antibody. Currently, general acquisition of a VHH using immunization of camelids is inconvenient from the standpoint of animal protection, cost and the process is time-consuming. Thus, a straightforward and efficient method for screening VHHs against a target molecule is required.

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Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements.

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In vitro display technologies such as mRNA and cDNA display are powerful tools to create and select functional peptides. However, in some cases, efficiency of mRNA-protein fusion is very low, which results in decreased library size and lower chance of successful selection. In this study, to improve mRNA-protein fusion efficiency, we prepared an mRNA display library of a protein with random N- and C-terminal coding regions consisting of 12 nucleotides (i.

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The first in vitro selection of binding peptides against artificial lipid membranes from a random peptide library using an in vitro display method (cDNA display) is reported. The selected peptide, LB-1, has both amphiphilic and cationic regions, and proteins fused to LB-1 can be immobilized on the liposome surface.

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RNA-protein interactions have a central role in the living world. In this article, we examined whether primitive peptides (30 residues) consisting of four types of amino acid (Gly, Ala, Asp, and Val) could interact with tRNA as a model of primitive RNAs in the RNA world. By in vitro selection of binding peptides using the cDNA display method, a characteristic peptide was selected from a random peptide library and assayed by electrophoretic mobility shift and pull-down assays.

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cDNA display is a powerful in vitro display technology used to explore functional peptides and proteins from a huge library by in vitro selection. In addition to expediting the in vitro selection cycle by using cDNA display, easy and rapid functional analysis of selected candidate clones is crucial for high-throughput screening of functional peptides and proteins. In this report, a versatile puromycin-linker employing an ultrafast photo-cross-linker, 3-cyanovinylcarbazole nucleoside, is introduced.

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Constrained peptides are an attractive class as affinity reagents or drug leads owing to their excellent binding properties. Many kinds of these peptides, such as cyclic peptides containing disulfide bridges, are found in nature or designed artificially by directed evolution. However, confirming the binding properties of the disulfide-rich peptides can be generally difficult, because of oxidative folding problems in the preparation steps.

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