Papers - USUI Kenji
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One-Pot Synthesis and Immobilization of Gold Nanoparticles Using Peptidyl Microbeads
Shuhei Yoshida,Koki Yoshida,Taichi Isozaki,Maho Oura,Makoto Ozaki,Takaaki Tsuruoka, Kenji Usui
Molecules 2025 30 ( 8 ) 1689 2025.4
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Development of detection system for lead ions in mixture solutions using UV-Vis measurements with peptide immobilized microbeads
Shuhei Yoshida, Koki Yoshida, Yoshio Hamada, Takaaki Tsuruoka, Kenji Usui
Scientific Reports 15 3249 2025.1
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Complex and Non-sequential Current Signatures of a β-Hairpin Peptide Confined in a Nanopore International coauthorship
Misa Yamaji, Mauro Chinappi, Blasco Morozzo della Rocca, Kenji Usui, Ryuji Kawano
Analytical Chemistry 97 ( 4 ) 2044 - 2051 2025.1
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Development of a CaCO3 Precipitation Method Using a Peptide and Microwaves Generated by a Magnetron Reviewed
Fumihiro Kayamori, Hiroyuki Togashi, Natsumi Endo, Makoto Ozaki, Kan Hirao, Yonejiro Arimoto, Ryuji Osawa, Takaaki Tsuruoka, Takahito Imai, Kin-ya Tomizaki, Tomohiro Umetani, Nobuhiro Nakanishi, Kenji Usui
Processes 12 ( 7 ) 1327 - 1327 2024.6
Publisher：MDPI AG
Microwave applications, such as microwave ovens and mobile phones, are ubiquitous and indispensable in modern society. As the utilization of microwave technology is becoming more widespread, the effects of microwaves on living organisms and physiological processes have received increased attention. This study aimed to investigate the effects of microwaves on calcium carbonate biomineralization as a model biochemical process. A magnetron oscillator was used to generate 2450 MHz microwaves because magnetrons are relatively inexpensive and widespread. We conducted transmission electron microscopy (TEM), atomic force microscopy (AFM), TEM-electron energy-loss spectroscopy (EELS), dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC) measurements to analyze the calcium carbonate precipitates. Our findings showed the formation of string-like precipitates of calcium carbonate upon microwave irradiation from one direction, similar to those obtained using a semiconductor oscillator, as reported previously. This implied that the distribution of the frequency had little effect on the morphology. Furthermore, spherical precipitates were obtained upon microwave irradiation from two directions, indicating that the morphology could be controlled by varying the direction of microwave irradiation. Magnetrons are versatile and also used in large-scale production; thus, this method has potential in medical and industrial applications.
DOI： 10.3390/pr12071327
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Effect of linearly polarized microwaves on nanomorphology of calcium carbonate mineralization using peptides Reviewed
Kenji Usui, Makoto Ozaki, Kan Hirao, Tsubasa Kosaka, Natsumi Endo, Shuhei Yoshida, Shin-ichiro Yokota, Yonejiro Arimoto, Ryuji Osawa, Nobuhiro Nakanishi, Kin-ya Tomizaki, Tomohiro Umetani, Fumihiro Kayamori
Scientific Reports 13 12027 2023.7
Microwaves are used for diverse applications such as mobile phones, ovens, and therapy devices. However, there are few reports on the effects of microwaves on diseases other than cancer, and on physiological processes. Here, we focused on CaCO3 mineralization as a model of biomineralization and attempted to elucidate the effect of microwaves on CaCO3 mineralization using peptides. We conducted AFM, ζ potential, HPLC, ICP-AES, and relative permittivity measurements. Our findings show that microwaves alter the nanomorphology of the CaCO3 precipitate, from sphere-like particles to string-like structures. Furthermore, microwaves have little effect on the mineralization when the mineralization ability of a peptide is high, but a large effect when the precipitation ability is low. Our findings may be applicable to not only the treatment of teeth and bones but also the development of organic–inorganic nanobiomaterials. This methodology can be expanded to other molecular/atomic reactions under various microwave conditions to alter reaction activity parameters.
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Construction of a Method to Design Fibril-Forming Peptides Applied to Biomaterials from 20 Beta-Sheet Peptides by Statistical Analysis Reviewed
Kazuya Iwata, Taisei Terao, Akira Takekawa, Tomohiro Umetani, Kenji Usui
Peptide Science 2022 141 - 142 2023.3
Authorship：Last author, Corresponding author
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De novo design of a nanopore for single-molecule detection that incorporates a β-hairpin peptide. Reviewed International coauthorship International journal
Keisuke Shimizu, Batsaikhan Mijiddorj, Masataka Usami, Ikuro Mizoguchi, Shuhei Yoshida, Shiori Akayama, Yoshio Hamada, Akifumi Ohyama, Kenji Usui, Izuru Kawamura, Ryuji Kawano
Nature nanotechnology 17 ( 1 ) 67 - 75 2022.1
The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a β-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a β-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.
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ウォッシャーディスインフェクターのすすぎ排水中に含まれる洗浄剤残留量の測定によるすすぎ性能評価 Reviewed
三軒隼人、藤田敏、川田原瑠勇、武川公、臼井健二、原田陽滋
医療機器学 91 315 - 332 2021.8
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Ozaki, M., Yoshida, S., Tsuruoka, T., Usui, K.
Chemical Communications 57 ( 6 ) 2021
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Ozaki, M., Imai, T., Tsuruoka, T., Sakashita, S., Tomizaki, K.-Y., Usui, K.
Communications Chemistry 4 ( 1 ) 2021
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Miyazaki, H., Hamada, Y., Takaishi, H., Minamino, Y., Ikeda, H., Mekata, H., Takaishi, M., Yamashita, K., Usui, K.
Analyst 145 ( 9 ) 3211 - 3216 2020.5
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Novel purification process for amyloid beta peptide(1-40) Invited Reviewed
Usui, K., Yokota, S.-I., Iwata, K., Hamada, Y.
Processes 8 ( 4 ) 2020
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Miyazaki, H., Takaishi, H., Ikeda, H., Ariumi, H., Hamada, Y., Yamashita, K., Usui, K.
Processes 8 ( 10 ) 2020
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Miyazaki, H., Samejima, Y., Iwata, K., Minamino, Y., Hikida, S., Ariumi, H., Ikeda, H., Hamada, Y., Yamashita, K., Usui, K.
International Journal of Molecular Sciences 21 ( 21 ) 2020
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Effect of tryptophan residues on gold mineralization by a gold reducing peptide Reviewed
Ozaki, M., Yoshida, S., Oura, M., Tsuruoka, T., Usui, K.
RSC Advances 10 ( 66 ) 2020
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Gold-Titania Nanocatalyst Generated by Mineralization Using Two Artificial Peptides with DNA Reviewed
Ozaki Makoto, Tomizaki Kin-Ya, Hamada Yoshio, Usui Kenji
JOURNAL OF PEPTIDE SCIENCE 24 S101 2018.9
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Kenji Usui, Shin-Ichiro Yokota, Makoto Ozaki, Shungo Sakashita, Takahito Imai, Kin-Ya Tomizaki
Protein and Peptide Letters 25 ( 1 ) 42 - 47 2018.4
Joint Work
Authorship：Lead author Publisher：Bentham Science Publishers B.V.
Background: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP-1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. Objectives: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. Methods: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). Results: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro-and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. Conclusion: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research.
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Yuki Tominaga, Kenji Usui, Akiyoshi Hirata, Hiro-O Ito, Kiyoshi Nokihara
Bioorganic and Medicinal Chemistry 26 ( 12 ) 3210 - 3216 2018.4
Joint Work
Authorship：Lead author Publisher：Elsevier Ltd
A fundamental method has been developed focusing on a facile and rapid examination of periodontal disease. Periodontal disease is an oral disease thought to affect 80% of adults, and early detection with treatment is desirable for the improvement of the quality of life. Unfortunately conventional methods are not consistent as the disease is caused by a number of undefined bacteria and detection relies on the skills of the dentist. Thus an objective detection system is required. We have performed an experiment on saliva using a novel biodetection system, designated PepTenChip®. A disease model for saliva was prepared using a specimen from a healthy subject and a mixture of hemoglobin (f-Hb) and lactate dehydrogenase (LDH), which is used as a periodontal disease marker protein with healthy saliva. PepTenChip® is a peptide microarray in which fluorescent labelled structured peptides are immobilized on a novel amorphous carbon substrate. Since the peptides used as capture molecules are fluorescently labelled, labeling of analytes is not necessary. The fluorescence intensity change before and after application of analytes are detected rather than the ON/OFF detection common to conventional microarrays using a set of antigen–antibody. The fluorescence intensity value changes according to the concentration of captured protein allowing the generation of protein fingerprint (PFP) and dendrograms. The present method does not rely on a “one to one” interaction, unlike conventional biodetection, and advantages can be envisaged in the case of an undefined or unknown cause of disease. The statistical analyses, such as multivariate analyses, allow classification of the type of proteins added in saliva as mimetics of disease. PepTenChip® system is useful and convenient for examination of periodontal disease in health care.
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Editorial: Organic-Inorganic Hybrid Materials and Their Applications Reviewed
Kin-ya Tomizaki, Yoshio Hamada, Kenji Usui
Protein & Peptide Letters 25 2 - 3 2018.4
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Peptides for Silica Precipitation: Amino Acid Sequences for Directing Mineralization Reviewed
Makoto Ozaki, Shungo Sakashita, Yoshio Hamada, Kenji Usui
Protein & Peptide Letters 25 ( 1 ) 15 - 24 2018.4
Joint Work
Publisher：Bentham Science Publishers B.V.
Background: Peptides are promising compounds for use in inorganic or organic-inorganic hybrid syntheses (mineralization) and offer several advantages over proteins. Meanwhile, silica-based nanomaterials have been extensively investigated for many years because of their potential application in a diverse range of technologies, including catalysis, sensing, separation, enzyme immobilization, and gene and drug delivery. Considerable progress has been made over the past decade in understanding the molecular mechanisms underpinning biosilicification and the biomimetic synthesis of patterned nanosilica using peptides. Objectives: This mini-review focuses on various peptide sequences, especially short peptide sequences (30 residues or less), for silica mineralization. Methods: We first briefly review early studies on silica mineralization using proteins to provide background information. This is followed by a discussion of promising peptide sequences and attempts to discern the relationship between amino acid sequence, their potential for mineralization, and the properties of the mineral product. Results: The synthetic control of silica mineralization using engineered proteins, such as recombinant silicateins and silaffins, was inspired by silica biomineralization by natural proteins from organisms (sponges, diatoms, and plants). Concurrently, several papers described the utility of well-structured protein assemblies as templates for silica mineralization. These template-directed syntheses of well-structured silica deposits were first conducted using natural proteins or protein assemblies such as collagen fibers and virus hollow protein tubes. Then we reviewed a selection of short peptides (30 residues or less) that had been successfully used for silica mineralization. Almost all peptides developed to date can be sorted by classification like proteins (synthetic control of silica mineralization or utility of templates for silica mineralization): the first class of peptides is used for peptide-directed synthesis, and the second is used for template-directed synthesis after the peptides have assembled and formed nanostructure such as fibers and tubes. The presented peptides were classified and arranged according to the classification. Additionally, we briefly introduced silica mineralization triggered by the combination of short silica-precipitating peptides and template molecules. Conclusion: In this mini-review we focused on various peptide sequences, especially short peptide sequences of 30 residues or less, designed for silica mineralization. The peptides have been used both for peptide-directed silica mineralization and for template-directed silica mineralization. The recent advances in peptide-driven mineralization reviewed here suggest that it will soon be possible to completely control the silica mineralization process using peptides. Mineralization systems using peptides will provide researchers with new tools for controlling various inorganic syntheses and the production of organic-inorganic materials for nanobiochemistry and materials chemistry research.
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Design of anti-alzheimer’s disease agents focusing on a specific interaction with target biomolecules Reviewed
Yoshio Hamada, Kenji Usui
Neuromethods 132 207 - 228 2018
Joint Work
Publisher：Humana Press Inc.
Alzheimer’s disease (AD) is the most common cause of dementia, characterized by progressive intellectual deterioration. Amyloid β peptide (Aβ), the main component of senile plaques in the brains of patients with AD, is formed from amyloid precursor protein (APP) by two processing enzymes. According to the amyloid hypothesis, a processing enzyme β-secretase (BACE1
β-site APP cleaving enzyme) that triggers Aβ formation in the rate-limiting first step of Aβ processing appears to be a promising molecular target for therapeutic intervention in AD. Many researchers have revealed BACE1 inhibitors for the AD treatment. Early BACE1 inhibitors were designed based on the first reported X-ray crystal structure, 1FKN, of a complex between recombinant BACE1 and inhibitor OM99-2. Although OM99-2 seemed to interact with BACE1-Arg235 side chain by hydrogen bonding, we found that a quantum chemical interaction, such as σ-π interaction or π-π stacking, plays a critical role in BACE1 inhibition mechanism. Moreover, we proposed a novel “electron-donor bioisostere” concept in drug discovery study and designed potent BACE1 inhibitors using this concept. -
Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides Reviewed
Sekiya, Y., Sakashita, S., Shimizu, K., Usui, K., Kawano, R.
Analyst 143 ( 15 ) 2018
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生体分子の挙動解析研究を目標としたマイクロ波照射システムの開発 ～ペプチドのバイオミネラリゼーションにおけるマイクロ波影響解析をモデルとして～ Reviewed
臼井健二, 富樫浩行, 圓東那津実, 尾崎誠, 有本米次郎, 裏鍛武史, 大沢隆二, 皆木幸一, 中西伸浩, 梅谷智弘
日本電磁波エネルギー応用学会論文誌 17 - 24 2017.12
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Non-Covalent Loading of Anti-Cancer Doxorubicin by Modularizable Peptide Self-Assemblies for a Nanoscale Drug Carrier Reviewed
Kin-ya Tomizaki, Kohei Kishioka, Shunsuke Kataoka, Makoto Miyatani, Takuya Ikeda, Mami Komada, Takahito Imai, Kenji Usui
MOLECULES 22 ( 11 ) 1916 2017.11
Joint Work
Authorship：Lead author Publisher：MDPI AG
We prepared nanoscale, modularizable, self-assembled peptide nanoarchitectures with diameters less of than 20 nm by combining beta-sheet-forming peptides tethering a cell-penetrating peptide or a nuclear localization signal sequence. We also found that doxorubicin (Dox), an anti-cancer drug, was non-covalently accommodated by the assemblies at a ratio of one Dox molecule per ten peptides. The Dox-loaded peptide assemblies facilitated cellular uptake and subsequent nuclear localization in HeLa cells, and induced cell death even at low Dox concentrations. This peptide nanocarrier motif is a promising platform for a biocompatible drug delivery system by altering the targeting head groups of the carrier peptides.
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DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity Reviewed
Kenji Usui, Arisa Okada, Shungo Sakashita, Masayuki Shimooka, Takaaki Tsuruoka, Shu-ichi Nakano, Daisuke Miyoshi, Tsukasa Mashima, Masato Katahira, Yoshio Hamada
MOLECULES 22 ( 11 ) 1991 2017.11
Joint Work
Authorship：Lead author Publisher：MDPI AG
The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca2+, and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.
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Fluorescent and luminescent fusion proteins for analyses of amyloid beta peptide aggregation Reviewed
Kenji Usui, Masayasu Mie, Takashi Andou, Hisakazu Mihara, Eiry Kobatake
JOURNAL OF PEPTIDE SCIENCE 23 ( 7-8 ) 659 - 665 2017.7
Joint Work
Authorship：Lead author Publisher：WILEY
The amyloid beta (A) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze A aggregation. A system was developed to monitor changes in luminescence that provides information about A aggregation. In the presence of monomeric A, the fusion protein exhibits higher luminescence intensity, and the luminescence intensity is diminished after aggregation of the fusion protein and A. In contrast, the fluorescence is sustained in the presence of A. In the absence of A, the fusion protein self-aggregates, and its luminescence and fluorescence are quenched, thus decreasing the background fluorescence and enhancing the detection of A inside and outside the cells. The ratio of the luminescence intensity to the fluorescence intensity would allow the aggregation degrees of A to be distinguished. This study would be a promising method for analyzing the aggregation state of a particular amyloid protein/peptide (monomer, oligomer, or fibril), as well as the distribution of the amyloid protein/peptide within and at the cell surface, by using a single fusion protein. Copyright (c) 2017 European Peptide Society and John Wiley & Sons, Ltd.
DOI： 10.1002/psc.3003
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Development of Peptide Microarray to Detect the Protein in Saliva for Periodontal Disease Test Reviewed
Yuki Tominaga, Kenji Usui, Akiyoshi Hirata, Atsushi Kitagawa, Hiro-O Ito and Kiyoshi Nokihara
Peptide Science 2016 131 - 132 2017.3
Joint Work
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A Systematic Study on Molecular Mechanism of Pore-Forming Peptides for Discovering Antimicrobial Medicine
Y. Sekiya, H. Watanabe, K. Usui, R. Kawano
Proceedings of MicroTAS 2016 595 - 596 2016.10
Joint Work
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Site-Specific Control of Silica Mineralization on DNA Using a Designed Peptide Reviewed
M. Ozaki, K. Nagai, H. Nishiyama, T. Tsuruoka, S. Fujii, T. Endoh, T. Imai, K.-y. Tomizaki, K. Usui
Chem. Commun. 52 4010 - 4013 2016.3
Joint Work
Authorship：Lead author
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Development of Designed Peptides with Secondary Structure for Use in Nanobiotechnology Invited Reviewed
K.Usui
Peptide Science 2015 5 - 8 2016.3
Single Work
Authorship：Lead author
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A Cell Microarray Format: A Peptide Release System Using a Photo-Cleavable Linker for Cell Toxicity and Cell Uptake Analysis. Reviewed
Usui K, Tomizaki KY, Mihara H
Methods in molecular biology (Clifton, N.J.) 1352 199 - 210 2016
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Site-specific control of multiple mineralizations using a designed peptide and DNA Reviewed
Kenji Usui, Makoto Ozaki, Aoi Yamada, Yoshio Hamada, Takaaki Tsuruoka, Takahito Imai, Kin-ya Tomizaki
NANOSCALE 8 ( 39 ) 17081 - 17084 2016
Publisher：ROYAL SOC CHEMISTRY
We have developed a site-specific method for precipitating multiple inorganic compounds using target DNA and a designed peptide consisting of a peptide nucleic acid (PNA) sequence and an inorganic compound-precipitating sequence. This system for controlled site-specific precipitation represents a powerful tool for use in nanobiotechnology and materials science.
DOI： 10.1039/c6nr03468c
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Site-specific control of silica mineralization on DNA using a designed peptide Reviewed
Makoto Ozaki, Kazuma Nagai, Hiroto Nishiyama, Takaaki Tsuruoka, Satoshi Fujii, Tamaki Endoh, Takahito Imai, Kin-ya Tomizaki, Kenji Usui
CHEMICAL COMMUNICATIONS 52 ( 21 ) 4010 - 4013 2016
Publisher：ROYAL SOC CHEMISTRY
We developed a site-specific method for precipitating inorganic compounds using organic compounds, DNA, and designed peptides with peptide nucleic acids (PNAs). Such a system for site-specific precipitation represents a powerful tool for use in nanobiochemistry and materials chemistry.
DOI： 10.1039/c5cc07870a
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ATP-Binding Peptide-Hydrogel Composite Synthesized by Molecular Imprinting on Beads Reviewed
Ayana Takata, Kenji Usui, Jun Matsui
Molecular Imprinting 3 65 - 70 2015.12
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A Cell Microarray Format: A Peptide Release System Using a Photo-Cleavable Linker for Cell Toxicity and Cell Uptake Analysis Reviewed
K. Usui, K.-y. Tomizaki, H. Mihara
Methods. Mol. Biol. 1352 199 - 210 2015.11
Joint Work
Authorship：Lead author
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Label and Label-Free Detection Techniques for Protein Microarrays. Reviewed International coauthorship
Syahir A, Usui K, Tomizaki KY, Kajikawa K, Mihara H
Microarrays (Basel, Switzerland) 4 ( 4 ) 228 - 244 2015.4
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Site-Specific Mineralization of Silica and Calcium on DNAs Using a Designed Peptide Reviewed
K. Usui, H. Nishiyama, A. Yamada, M. Ozaki, T. Tsuruoka, K.-y. Tomizaki
Peptide science 2014 325 - 326 2015.3
Joint Work
Authorship：Lead author
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Control of guanine-rich DNA secondary structures depending on the protease activity using a designed PNA peptide Reviewed
Kenji Usui, Arisa Okada, Keita Kobayashi, Naoki Sugimoto
ORGANIC & BIOMOLECULAR CHEMISTRY 13 ( 7 ) 2022 - 2025 2015
Publisher：ROYAL SOC CHEMISTRY
We constructed a regulation system for DNA secondary structure formation of G-rich sequences using a designed PNA peptide exhibiting an on-to-off switching functionality, depending on the protease activity. This study introduces the new concept of a simple and powerful system for regulating quadruplex-related important biological events.
DOI： 10.1039/c4ob02535k
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Cellular differentiation assessments by measuring the degree of cellular internalization and membrane adsorption using designed peptides Reviewed
Kenji Usui, Takuya Kikuchi, Kunio Kikuchi, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 24 ( 17 ) 4129 - 4131 2014.9
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
We demonstrate examples of cellular differentiation assessments, including cellular neurite outgrowth and fat cell maturation, by measuring the degree of membrane adsorption or cellular internalization using designed peptides. Because changes in the cellular membrane and cytosol during differentiation were shown to influence membrane adsorption and cellular internalization, we could successfully evaluate the extent of differentiation simply like stain indicators. (C) 2014 Elsevier Ltd. All rights reserved.
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A Designed Small Protein For Controlling Site-Specific Mineralization Of Silica And Calcium On Dnas Reviewed
Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Aoi Yamada, Makoto Ozaki, Takaaki Tsuruoka, Kin-ya Tomizaki
PROTEIN SCIENCE 23 143 - 143 2014.7
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Site-Specific Control of Silica Mineralization on DNA Using Designed Peptides Reviewed
K. Usui, K. Nagai, H. Nishiyama, A. yamada, T. Tsuruoka, S. Fujii, K.-y. Tomizaki
Peptide Science 2013 2415 - 2416 2014.3
Joint Work
Authorship：Lead author
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Peptides targeting G-quadruplex structures Reviewed
Kenji Usui, Arisa Okada
Chemical Biology of Nucleic Acids: Fundamentals and Clinical Applications 459 - 475 2014.1
Publisher：Springer Berlin Heidelberg
Research in recent decades has revealed that some DNA and RNA secondary structures modulate a variety of cellular events. One secondary structure, the Guanine(G)-quadruplex, also regulates various cellular events that are mostly related to serious diseases. Systems capable of controlling DNA and RNA G-quadruplex structures would therefore be useful for the modulation of various cellular events to produce biological effects. Because of their biological importance, many G-quadruplex-targeting compounds have been described. However, the next generation of targeting molecules should exhibit increased G-quadruplex sequence specificity, a higher structure-inducing or -collapsing ability, and a greater degree of functionality, including on–off switches of binding ability and cellular penetration. Peptides might be good candidates for these next-generation Gquadruplex- targeting molecules due to the following advantages: (1) their easy design and synthesis, (2) their ability to mimic protein–G–quadruplex interactions, (3) the possibility of employing artificial amino acids in addition to naturally occurring amino acids, and (4) the ability to combine G-quadruplex-binding sequences with other functional sequences. Accordingly, several peptide-based compounds, such as furan-based cyclic peptides, PNA-conjugated peptides, and small molecule-peptide conjugates, have been developed. In this chapter, we introduce all these peptide ligands and describe most of the approaches for targeting G-quadruplex structures. We then conclude that peptides are among the most promising functional ligands for G-quadruplexes to control various biological events in next-generation approaches.
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Control of Site-Specific Silica Precipitation Using PNA Peptides and DNAs Reviewed
K. Usui, H. Nishiyama, K. Nagai, T. Tsuruoka, S. Fujii, K.-y. Tomizaki
Peptides 2013 162 - 163 2013.12
Joint Work
Authorship：Lead author
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Designed Peptide Libraries for Cell Analyzing Microarrays Reviewed
H. Mihara, K. Usui, H. Tsutsumi, K. Nokihara
Peptides 2013 22 - 23 2013.12
Joint Work
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Peptide-DNA Hybrid G-Quadruplex Structures for Regulation of Protein Expression Depending on Protease Activity Reviewed
A. Okada, M. Taniguchi, K. Usui, N. Sugimoto
Peptides 2013 180 - 181 2013.12
Joint Work
Authorship：Lead author
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Systematic screening of the cellular uptake of designed alpha-helix peptides Reviewed
Kenji Usui, Takuya Kikuchi, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY 21 ( 9 ) 2560 - 2567 2013.5
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
The cellular penetration (CP) activity of functional molecules has attracted significant attention as one of the most promising new approaches for drug delivery. In particular, cell-penetrating peptides (CPPs) have been studied extensively in cellular engineering. Because there have been few large-scale systematic studies to identify peptide sequences with optimal CP activity or that are suitable for further applications in cell engineering, such as cell-specific penetration and cell-selective culture, we screened and compared the cellular uptake (CU) activity of 54 systematically designed a-helical peptides in HeLa cells. Furthermore, the CU activity of 24 designed peptides was examined in four cell lines using a cell fingerprinting technique and statistical approaches. The CU activities in various cells depended on amino acid residues of peptide sequences as well as charge, a-helical content and hydrophobicity of the peptides. Notably, the mutation of a single residue significantly altered the CU ability of a peptide, highlighting the variability of cell uptake mechanisms. Moreover, these results demonstrated the feasibility of cell-selective culture by conducting cell-selective permeation and death in cultures containing two cell types. These studies may lead to further peptide library design and screening for new classes of CPPs with useful functions. (C) 2013 Elsevier Ltd. All rights reserved.
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A peptide release system using a photo-cleavable linker in a cell array format for cell-toxicity analysis Invited Reviewed
Takashi Kakiyama, Kenji Usui, Kin-ya Tomizaki, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
POLYMER JOURNAL 45 ( 5 ) 525 - 539 2013.5
Joint Work
Authorship：Lead author Publisher：NATURE PUBLISHING GROUP
We constructed a novel peptide-array format system for cellular toxicity analysis. In this system, a peptide was immobilized on a conventional 96-well plate bottom via a photo-cleavable linker. Once UV light irradiated the desired wells, the peptide was released from the bottom. As a result, the cytotoxic behavior of the peptide could be monitored. Immobilization and light-irradiation conditions were optimized. The immobilized peptide showed no cytotoxicity; therefore, the cells could be cultured on the peptide-immobilizing plate from the beginning of the experiment. Cell-toxicity assays with this system for three cell types were performed. All cell types showed similar to 25% lowering of viability with the photo-released 5-(and-6)-carboxytetramethylrhodamine (TMR)-GKLAKLAKKLAKLAKKLAKLAKGC (TMR-KLA-C) peptide compared with the non-coated plate. This relative toxicity nearly corresponded to that of similar to 10 mu M TMR-KLA-C in solution, and we found that the released peptide concentration per well was similar to 10 mu M at 60 min irradiation. Throughout this study, we successfully immobilized peptides via the photo-cleavable linker, released them by UV irradiation spatiotemporally and conducted the cell-toxicity assay. This study implies that the peptide photo-releasing array system will allow the realization of high-throughput cell arrays for cellomics analyses and cell-based phenotypic drug screenings.
DOI： 10.1038/pj.2013.20
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Construction of Artificial Peptides for Control of DNA G-quadruplex Structures Depending on Protease Activity toward Regulation of Protein Expression Reviewed
Arisa Okada, Kenji Usui, Mai Taniguchi, Keita Kobayashi, Naoki Sugimoto
Peptide Science 2012 285 - 286 2013.3
Joint Work
Authorship：Lead author
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A PNA Peptide for the Control of Site-Specific Silica Precipitation on DNA Reviewed
Kenji Usui, Kazuma Nagai, Hiroto Nishiyama, Takaaki Tsuruoka, Satoshi Fujii
Peptide Science 2012 375 - 376 2013.3
Joint Work
Authorship：Lead author
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A novel array format for monitoring cellular uptake using a photo-cleavable linker for peptide release Reviewed
Kenji Usui, Takuya Kikuchi, Kin-ya Tomizaki, Takashi Kakiyama, Hisakazu Mihara
CHEMICAL COMMUNICATIONS 49 ( 57 ) 6394 - 6396 2013
Publisher：ROYAL SOC CHEMISTRY
We developed a novel peptide array format incorporating a photo-cleavable linker for monitoring cellular uptake. Model peptides were successfully immobilised via the photo-cleavable linker onto conventional plates and could be released spatiotemporally using UV irradiation. Incorporation of confocal microscopy allowed for detailed real-time monitoring of cellular internalisation of peptides.
DOI： 10.1039/c3cc41632a
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Screening of Designed Peptides for Binders to a DNA G-Quadruplex Structure Reviewed
N. Matsui, K. Kobayashi, K. Usui
Peptide Science 2011 2011 315 - 316 2012.3
Joint Work
Authorship：Lead author
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De Novo Designed Nitric Oxide Sensor Protein consisted of Iron Complex-Peptide Conjugate Reviewed
H. Miyazaki, K. Usui, S. Fujii
Peptide Science 2011 2011 303 - 304 2012.3
Joint Work
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Development of PNA-Peptides Controlling DNA G-Quadruplex Structures Depending on Protease Activity Reviewed
K. Usui, K. Kobayashi, N. Sugimoto
Peptide Science 2011 2012 315 - 316 2012.3
Joint Work
Authorship：Lead author
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Cell fingerprint patterns using designed alpha-helical peptides to screen for cell-specific toxicity Reviewed
Kenji Usui, Takashi Kakiyama, Kin-ya Tomizaki, Masayasu Mie, Eiry Kobatake, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 21 ( 21 ) 6281 - 6284 2011.11
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
We conducted cell-based cytotoxicity screening of a 101-membered alpha-helical peptide library using cell fingerprints (CFPs). The CFP data suggested that there is a relationship between cytotoxicity and peptide characteristics, such as hydrophobicity, charge, and amino acid composition. In spite of the small size of the library used in this study, several peptides demonstrated cell-specific toxicity. The strategy of combining a designed peptide library with CFP thus shows real promise for peptide-based screening with cells. (C) 2011 Elsevier Ltd. All rights reserved.
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Thermodynamic stability of Hoogsteen and Watson-Crick base pairs in the presence of histone H3-mimicking peptide Reviewed International coauthorship
Smritimoy Pramanik, Kaori Nakamura, Kenji Usui, Shu-ichi Nakano, Sarika Saxena, Jun Matsui, Daisuke Miyoshi, Naoki Sugimoto
CHEMICAL COMMUNICATIONS 47 ( 10 ) 2790 - 2792 2011
Publisher：ROYAL SOC CHEMISTRY
We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.
DOI： 10.1039/c0cc05776b
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Use of a designed peptide library to screen for binders to a particular DNA G-quadruplex sequence Reviewed
Keita Kobayashi, Noriko Matsui, Kenji Usui
Journal of Nucleic Acids 2011 572873 2011
We demonstrated a method to screen for binders to a particular G-quadruplex sequence using easily designed short peptides consisting of naturally occurring amino acids and mining of binding data using statistical methods such as hierarchical clustering analysis (HCA). Despite the small size of the library used in this study, candidates of specific binders were identified. In addition, a selected peptide stabilized the G-quadruplex structure of a DNA oligonucleotide derived from the promoter region of the protooncogene c-MYC. This study illustrates how a peptide library can be designed and presents a screening guideline for construction of G-quadruplex binders. Such G-quadruplex peptide binders could be functionally modified to enable switching, cellular penetration, and organelle-targeting for cell and tissue engineering. © 2011 Keita Kobayashi et al.
DOI： 10.4061/2011/572873
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PNA-Peptide Conjugates for Regulation of DNA and RNA G-Quadruplex Structures Depending on a Particular Protease Concentration Reviewed
Kenji Usui, Keita Kobayashi, Naoki Sugimoto
Peptides 2010 2010 608 - 609 2011
Joint Work
Authorship：Lead author
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Nanomolar aggregation of amyloid-beta peptides covalently and site-specifically modified by cholesterol oxidation products Reviewed International coauthorship
Kenji Usui, Evan T. Powers, Johan F. Paulsson, Sarah J. Siegel & Jeffery W. Kelly
Peptides 2008 2008 588 - 589 2011
Joint Work
Authorship：Lead author
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Fluorescent and Luminescent Fusion Proteins for Detection of Amyloid Beta Peptide Localization and Aggregation Reviewed
Kenji Usui, Masayasu Mie, Takashi Andou, Naoki Sugimoto, Hisakazu Mihara, Eiry Kobatake
Peptides 2010 2010 488 - 489 2011
Joint Work
Authorship：Lead author
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Designed Peptide Libraries for Protein and Cell analyses Reviewed
K. Kikuchi, K. Usui, K.-Y. Tomizaki, T. Takahashi, H. Mihara
Peptide Science 2010 2010 21 - 21 2011
Joint Work
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Nanomolar Aggregation and Neurotoxicity of Amyloid beta Peptides with Site-Specific Modification of Oxidized Cholesterol Reviewed International coauthorship
K. Usui, J. D. Hulleman, J. F. Paulsson, S. J. Siegel, E. T. Powers, J. W. Kelly
Peptide Science 2010 2010 169 - 169 2011
Joint Work
Authorship：Lead author
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A Novel Nitric Oxide Sensor Using Fluorescent Peptides Attached to Iron Complexes Reviewed
Hiroshi Miyazaki, Kenji Usui, Satoshi Fujii
Peptides 2011 2011 224 - 255 2011
Joint Work
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A PNA Peptide Inducing DNAs to Form G-Quadruplex Structures Depending on a Protease Activity Reviewed
Kenji Usui, Keita Kobayashi, Naoki Sugimoto
Peptides 2011 2011 152 - 153 2011
Joint Work
Authorship：Lead author
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Construction of Detection System for Amyloid beta Peptide Localization and Aggregation Using Fluorescent and Luminescent Fusion Proteins Reviewed
Kenji Usui, Masayasu Mie, Takashi Andou, Naoki Sugimoto, Hisakazu Mihara, Eiry Kobatake
Peptide Science 2009 2009 111 - 112 2010
Joint Work
Authorship：Lead author
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Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity Reviewed International coauthorship
Kenji Usui, John D. Hulleman, Johan F. Paulsson, Sarah J. Siegel, Evan T. Powers, Jeffery W. Kelly
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 ( 44 ) 18563 - 18568 2009.11
Publisher：NATL ACAD SCIENCES
Accumulation of amyloid beta-peptide (A beta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between A beta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase A beta amyloidogenicity. Here, we synthesized A beta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified A beta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from A beta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified A beta under identical conditions and at the same concentration. Our results show that A beta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of A beta.
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A designed peptide chip: Protein fingerprinting technology with a dry peptide array and statistical data mining Reviewed
Kenji Usui, Kin-Ya Tomizaki, Hisakazu Mihara
Methods in Molecular Biology 570 273 - 284 2009
There has recently been increased interest in the potential for microarray technologies to study protein networks in a whole cell system within a single experiment. Protein-detecting microarrays are composed of numerous agents immobilized within a tiny area on solid surfaces to capture targeted proteins and to detect interactions in a high-throughput fashion. In this chapter, in order to extend the usability of peptide microarrays, we describe a novel dry peptide microarray format to obtain protein fingerprint (PFP) data sets and a statistical PFP data manipulation technique to quantitatively analyze targeted proteins. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.
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Screening of alpha-helical peptide ligands controlling a calcineurin-phosphatase activity Reviewed
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 17 ( 1 ) 167 - 171 2007.1
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
In this paper, we describe an application of 202-membered fluorescently labeled peptide library designed to take an alpha-helix secondary structure. As a proof-of-concept experiment, a calmodulin (CaM)/calcineurin (Cn) pair was chosen to screen alpha-helical peptide ligands that tightly bind to CaM and also control enzymatic functions of Cn. Three peptides were successfully selected from the library by assaying Cn-phosphatase activities and peptide-CaM interactions (dual check process). The strategy using a designed peptide. library shows real promise as a peptide-based high-throughput screening system. (c) 2006 Elsevier Ltd. All rights reserved.
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Interactions between peptides containing nucleobase amino acids and T7 phages displaying S-cerevisiae proteins Reviewed
Sinya Watanabe, Kin-ya Tomizaki, Tsuyoshi Takahashi, Kenji Usui, Kotaro Kajikawa, Hisakazu Mihara
BIOPOLYMERS 88 ( 2 ) 131 - 140 2007
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Protein-fingerprint data mining of a designed alpha-helical peptide array Reviewed
Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
MOLECULAR BIOSYSTEMS 2 ( 9 ) 417 - 420 2006.9
Publisher：ROYAL SOC CHEMISTRY
The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins. The data generated from protein fingerprints with an a-helical peptide array were analyzed using several statistical methods such as hierarchical clustering analysis and principal component analysis to discriminate target proteins.
DOI： 10.1039/b608875a
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A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system Reviewed
K Usui, K Tomizaki, T Ohyama, K Nokihara, H Mihara
MOLECULAR BIOSYSTEMS 2 ( 2 ) 113 - 121 2006.2
Publisher：ROYAL SOC CHEMISTRY
A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure tinder dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-Of-Use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an a-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These Studies imply that the dry peptide array system is a promising too] for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.
DOI： 10.1039/b514263r
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A PNA-DNA hybridization chip approach for the detection of beta-secretase activity Reviewed
S Sano, KY Tomizaki, K Usui, H Mihara
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 16 ( 3 ) 503 - 506 2006.2
Publisher：PERGAMON-ELSEVIER SCIENCE LTD
Developed was the addressable chip technology based on the PNA-DNA complementary hybridization equipped with short seven-mer PNA-encoded peptides that can be a versatile scaffold to monitor on-chip immunoassays. We also developed and validated a methodology to perform beta-secretase enzyme assay with a highly sensitive fashion, resulting that a peptide substrate tethering dual fluorescent probes allowed us to detect beta-secretase activity 10 times more sensitively than assays in solution. (c) 2005 Elsevier Ltd. All rights reserved.
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Designed peptide microarrays for protein detection and characterization Reviewed
Kenji Usui, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 731 - + 2006
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High throughput preparation of peptide arrays containing two fluorescent byes focusing on practical protein detection systems Reviewed
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Yasuo Oka, Kenji Usui, Hisakazu Mihara
UNDERSTANDING BIOLOGY USING PEPTIDES 738 - + 2006
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Anomalous reflection of gold applicable for a practical protein-detecting chip platform Reviewed
S Watanabe, K Usui, K Tomizaki, K Kajikawa, H Mihara
MOLECULAR BIOSYSTEMS 1 ( 5-6 ) 363 - 365 2005.12
Publisher：ROYAL SOC CHEMISTRY
A simple, convenient and label-free fiber optic detection system based on the characteristic property, 'anomalous reflection (AR)' of gold was developed and preliminary experiments showed that the AR signals were sensitive enough to monitor protein-peptide interactions on solid surfaces.
DOI： 10.1039/b513075c
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Kenji Usui, Tetsunori Ojima, Kin-Ya Tomizaki, Hisakazu Mihara
Nanobiotechnology 1 ( 2 ) 191 - 199 2005.11
For the realization of a practical high-throughput protein detection and analysis system, a novel peptide array has been constructed using a designed glycopeptide model library with an α-helical secondary structure. This study will contribute the increment of the diversity of such an array system and the application to focused proteomics and ligand screening by effective detection of sugar-binding proteins. Fluorescent glycopeptides with an α-helix, a β-strand, or a loop structure were designed initially to select a suitable scaffold for the detection of a model protein. After selection of the α-helical structure as the best scaffold, a small model library with various saccharides was constructed to have charge and hydrophobicity variations in the peptide sequences. When various sugar-binding proteins were added to the peptide library array, the fluorescent peptides showed different responses in fluorescence intensities depending on their sequences as well as saccharides. The patterns of these responses could be regarded as "protein fingerprints" (PFPs), which are able to establish the identities of the target proteins. The resulting PFPs reflected the recognition properties of the proteins. Furthermore, statistical data analysis from obtained PFPs was performed using a cluster analysis. The PFPs of sugar-binding proteins were clustered successfully depending on their families and binding properties. These studies demonstrate that arrays with glycopeptide libraries based on designed structures can be promising tools to detect and analyze the target proteins. Designed peptides with functional groups such as sugars will play roles as the capturing agents of high-throughput protein nano/micro arrays for focused proteomics and ligand screening studies. Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever are reserved.
DOI： 10.1385/NBT:1:2:191
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Difference in self-assembling morphology of peptide nanorings Reviewed
H Okamoto, T Yamada, H Miyazaki, T Nakanishi, K Takeda, K Usui, Obataya, I, H Mihara, H Azehara, W Mizutani, K Hashimoto, H Yamaguchi, Y Hirayama
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 11 ) 8240 - 8248 2005.11
Publisher：JAPAN SOC APPLIED PHYSICS
We synthesized the peptide nanorings of cyclo[-(D-Ala-L-Gln)(3)], cyclo[-(D-CyS-L-Gln)(3)], CyC10[-D-Cys-L-HiS-D-Ala-L-Asn-Gly-L-Gln-1 and Cyc1o[-(L-Gln)(5)], and studied the way in which the difference in the type and/or number of component amino acid residues changes the self-assembling morphology of the nanorings on gold substrates by atomic force microscopy. The study revealed that CyClo[-(D-Ala-L-Gln)(3)] formed nanotube bundles through inter-ring hydrogen bonds, while the nanorings of CyC10[-(D-CyS-L-Gln)3] adhered to the gold surface directly due to the high affinity of thiol to gold. In contrast, a random amino acid sequence of cyclo[-D-CyS-L-HiS-D-Ala-L-Asn-GlY-L-Gln-] resulted in many isolated nanotubes, which were first observed in the present study. While the D,L-peptide nanotubes have very straight forms, the homo-L-peptide of cyclo[-(L-Gln)(5)] formed interesting randomly branching nanotubes that were entwined and grew on the substrate. Scanning tunneling microscopy was also performed and high-resolution images of both the peptide nanotubes and the nanotube bundles were obtained.
DOI： 10.1143/JJAP.44.8240
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IR study on stacking manner of peptide nanorings in peptide nanotubes Reviewed
Y Nagai, T Nakanishi, H Okamoto, K Takeda, Y Furukawa, K Usui, H Mihara
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS 44 ( 10 ) 7654 - 7661 2005.10
Publisher：JAPAN SOC APPLIED PHYSICS
We here report our theoretical as well as experimental studies on the stacking manner of peptide nanorings (PNRs) in peptide nanotubes (PNTs). We focus on the molecular vibrations of N-H and C=O stretching modes and discuss this subject via their infrared (IR) spectroscopy, because PNTs are formed by the inter-ring H bonds between the adjacent PNRs via -N-H(...)O=C-. Symmetry analysis based on group theory reveals that parallel stacking causes two IR active modes in these molecular vibrations while three modes are active in the antiparallel stacking. This difference in the number of IR active modes is determined only by the stacking manner and not by the number of amino acid residues composing the PNRs. By using two typical PNRs Of cyclo[-((L)-Gln-D-Ala)(3)] and cyclo[-((L)-Gln-(D)-Ala)(4)], we further studied the favorable stacking manners of PNRs via IR observation. Our IR experiments as well as the ab initio energetics show that the former PNRs create a PNT by stacking themselves in parallel while the latter PNRs do so by stacking themselves in an antiparallel manner.
DOI： 10.1143/JJAP.44.7654
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Protein-detecting microarrays: Current accomplishments and requirements Reviewed
Kin-Ya Tomizaki, Kenji Usui, Hisakazu Mihara
ChemBioChem 6 ( 6 ) 782 - 799 2005.5
Joint Work
The sequencing of the human genome has been successfully completed and offers the chance of obtaining a large amount of valuable information for understanding complex cellular events simply and rapidly in a single experiment. Interestingly, in addressing these proteomic studies, the importance of protein-detecting microarray technology is increasing. In the coming few years, microarray technology will become a significantly promising and indispensable research/diagnostic tool from just a speculative technology. It is clear that the protein-detecting microarray is supported by three independent but strongly related technologies (surface chemistry, detection methods, and capture agents). Firstly, a variety of surface-modification methodologies are now widely available and offer site-specific immobilization of capture agents onto surfaces in such a way as to keep the native conformation and activity. Secondly, sensitive and parallel detection apparatuses are being developed to provide highly engineered microarray platforms for simultaneous data acquisition. Lastly, in the development of capture agents, antibodies are now probably the most prominent capture agents for analyzing protein abundances. Alternative scaffolds, such as phage-displayed antibody and protein fragments, which provide the advantage of increasing diversity of proteinic capture agents, however, are under development. An approach involving recombinant proteins fused with affinity tag(s) and coupled with a highly engineered surface chemistry wilt provide simple production protocols and specific orientations of capture agents on the -microarray formats. Peptides and other small molecules can be employed in screening highly potent ligands as well as in measuring enzymatic activities. Protein-detecting microarrays supported by the three key technologies should contribute in accelerating diagnostic/biological research and drug discovery. © 2005 Wiley-VCH Verlag GmbH &
Co. KGaA. -
Studies on Fluorescent Dyes and Surface Chemistry focusing on Practical Peptide Array Preparation Reviewed
NOKIHARA Kiyoshi, USUI Kenji, YONEMURA Koichi, OHYAMA Takafumi, OKA Yasuo, TOMIZAKI Kin‐ya, MIHARA Hisakazu
Pept Sci 2004 145 - 148 2005.3
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Optimization of peptide arrays on chip as novel practical protein characterization system Reviewed
Kiyoshi Nokihara, Takafumi Ohyama, Koichi Yonemura, Kenji Usui, Kin-ya Tomizaki, Hisakazu Mihara
Peptides 2004, Proceedings 176 - 177 2005
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Protein-detection microarrays using structure-based designed peptide libraries Reviewed
Kenji Usui, Tetsunori Ojima, Masato Suzuki, Sin-ya Watanabe, Kin-ya Tomizaki, Kiyoshi Nokihara, Hisakazu Mihara
Peptides 2004, Proceedings 401 - 402 2005
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Peptide Arrays with Designed Secondary Structures for Protein Characterization Using Fluorescent Fingerprint Patterns. Reviewed
臼井健二
Biopolymers ( 76 ) 129 - 139 2004.11
Single Work
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Peptide arrays with designed alpha-helical structures for characterization of proteins from FRET fingerprint patterns Reviewed
Kenji Usui, Mizuki Takahashi, Kiyoshi Nokihara, Hisakazu Mihara
MOLECULAR DIVERSITY 8 ( 3 ) 209 - 218 2004.9
Publisher：SPRINGER
A practical high-throughput protein detection system is described, based on synthetic peptide arrays consisting of designed alpha-helical peptides, detected by fluorescence resonance energy transfer (FRET). Initially a model alpha-helical peptide known to interact with a structured protein, calmodulin, was selected to establish the strategy for high-throughput detection. In comparison to peptides with a single probe, a much higher FRET response has been observed with two fluorescent probes (7-diethylaminocoumarin-3-carboxylic acid and 5(6)-carboxy-fluorescein) at both termini of the synthetic peptides. To establish a reproducible high-throughput detection system, peptides were also immobilized onto a solid surface for detection of the target proteins. A small library of 112 different peptides was constructed, based on a model of the alpha-helical peptide with systematic replacement of residues carrying specific charges and/or hydrophobicities. The library was used to effectively characterize various proteins, giving their own 'protein fingerprint' patterns. The resulting 'protein fingerprints' correlate with the recognition properties of the proteins. The present microarray with designed synthetic peptides as the capturing agents is promising for the development of protein detection chips.
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Development of a practical protein-chip using designed synthetic peptide-arrays Reviewed
K Nokihara, T Ohyama, K Usui, K Yonemura, K Tomizaki, H Mihara
KOBUNSHI RONBUNSHU 61 ( 10 ) 523 - 532 2004
Publisher：SOC POLYMER SCIENCE JAPAN
Novel high-throughput technologies, which can replace conventional electrophoresis and mass spectroscopic analyses, are in great demand for understanding the structure-function relationships of proteins. For a practical protein-detection system, arrays with immobilized designed synthetic peptides have been constructed. Peptides were labeled with fluorescent dyes in order to achieve high sensitivity. Two different types of prototype-arrayer have been constructed: one has a micro-dispensing system and the other a spot-printing system. Combinatorial peptide libraries, which consisted of beta-loop, beta-strand and alpha-helical peptides, were constructed by improved highly efficient solid-phase synthesis. Labeled peptides were covalently immobilized on solid supports such as precision glass plates. Various proteins were characterized with these peptide-arrays using a fluorescent scanner to give the "protein fingerprint" which is characteristic of the individual proteins. The results of the present prototype system demonstrate the practicality of protein-chips as a new generation of biochips.
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Development of Peptide-Chips Focusing on a High Throughput Protein-Detection System Reviewed
Kiyoshi Nokihara, Takafumi Ohyama, Kenji Usui, Koichi. Yoneyama, Mizuki Takahashi, Hisakazu Mihara
Innovation and Perspectives in Solid Phase Synthesis & Combinatorial Libraries 83 - 88 2004
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Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns Reviewed
K Usui, T Ojima, M Takahashi, K Nokihara, H Mihara
BIOPOLYMERS 76 ( 2 ) 129 - 139 2004
Publisher：JOHN WILEY & SONS INC
To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, a-helix, or P-strand structures. Here, we describe the overview of the reported deigned peptide arrays with loop and a-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of a-helix peptide, the response of a peptide with fluorescent resonance energy tran, fer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model pcptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips. (C) 2004 Wiley Periodicals, Inc.
DOI： 10.1002/bip.10568
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Peptide microarrays using structure-based peptide libraries for protein chips Reviewed
Kenji Usui, Mizuki Takahashi, Tetsunori Ojima, Masato Suzuki, Kiyoshi Nokihara, Eiichi Tamiya, Hisakazu Mihara
Peptide Revolution: Genomics, Proteomics & Therapeutics 258 - 259 2004