Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：International academic award (Japan or overseas)  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：International academic award (Japan or overseas)  Country：Japan

Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Plant Cell  

DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined 2 proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single-base substitution (SBS) mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation data set. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1-binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

DOI： 10.1093/plcell/koae089

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：EMBO Journal  

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.

DOI： 10.1038/s44318-024-00036-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Methods in Enzymology  

Peptide ligation chemistries have revolutionized the synthesis of proteins with site-specific modifications or proteomimetics through assembly of multiple peptide segments. In order to prepare polypeptide chains consisting of 100–150 amino acid residues or larger generally assembled from three or more peptide segments, iterative purification process that decreases the product yield is usually demanded. Accordingly, methodologies for one-pot peptide ligation that omit the purification steps of intermediate peptide segments have been vigorously developed so far to improve the efficiency of chemical protein synthesis. In this chapter, we first outline the concept and recent advances of one-pot peptide ligation strategies. Then, the practical guideline for the preparation of peptide segments for one-pot peptide ligation is described with an emphasis on diketopiperazine thioester synthesis. Finally, we disclose the explicit protocols for one-pot four segment ligation via repetitive deprotection of N-terminal thiazolidine by a 2-aminobenzamide type aldehyde scavenger.

DOI： 10.1016/bs.mie.2024.04.020

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.21203/rs.3.rs-4275898/v1

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nucleic Acids Research  

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

DOI： 10.1093/nar/gkad754

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nucleic Acids Research  

Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such libraries with over 1012-1013 diversity. Particularly, the protein-puromycin linker (PuL)/mRNA complex formation yield is determining for preparing the libraries. However, how mRNA sequences affect the complex formation yield remains unclear. To study the effects of N-terminal and C-terminal coding sequences on the complex formation yield, puromycin-attached mRNAs containing three random codons after the start codon (32768 sequences) or seven random bases next to the amber codon (6480 sequences) were translated. Enrichment scores were calculated by dividing the appearance rate of every sequence in protein-PuL/mRNA complexes by that in total mRNAs. The wide range of enrichment scores (0.09-2.10 for N-terminal and 0.30-4.23 for C-terminal coding sequences) indicated that the N-terminal and C-terminal coding sequences strongly affected the complex formation yield. Using C-terminal GGC-CGA-UAG-U sequences, which resulted in the highest enrichment scores, we constructed highly diverse libraries of monobodies and macrocyclic peptides. The present study provides insights into how mRNA sequences affect the protein/mRNA complex formation yield and will accelerate the identification of functional peptides and proteins involved in various biological processes and having therapeutic applications.

DOI： 10.1093/nar/gkad555

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Language：English   Publishing type：Research paper (scientific journal)  

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

DOI： 10.3390/molecules28093655

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41589-022-01178-1

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DOI： 10.1093/nar/gkac1082

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nucleic acids research  

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

DOI： 10.1093/nar/gkac1082

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nar/gkac1082

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DOI： 10.1093/nar/gkac1082

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Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N',N'-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

DOI： 10.1002/anie.202206240

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Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Life Science Alliance  

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are useful for patients’ treatment of the coronavirus disease 2019 (COVID-19). We report here affinity maturation of monobodies against the SARS-CoV-2 spike protein and their neutralizing activity against SARS-CoV-2 B.1.1 (Pango v.3.1.14) as well as four variants of concern. We selected matured monobodies from libraries with multi-site saturation mutagenesis on the recognition loops through in vitro selection. One clone, the C4-AM2 monobody, showed extremely high affinity (KD < 0.01 nM) against the receptor-binding domain of the SARS-CoV-2 B.1.1, even in monomer form. Furthermore, the C4-AM2 monobody efficiently neutralized the SARS-CoV-2 B.1.1 (IC50 = 46 pM, 0.62 ng/ml), and the Alpha (IC50 = 77 pM, 1.0 ng/ml), Beta (IC50 = 0.54 nM, 7.2 ng/ml), Gamma (IC50 = 0.55 nM, 7.4 ng/ml), and Delta (IC50 = 0.59 nM, 8.0 ng/ml) variants. The obtained monobodies would be useful as neutralizing proteins against current and potentially hazardous future SARS-CoV-2 variants.

DOI： 10.26508/lsa.202101322

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cca.2022.04.313

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bio-protocol  

Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 1013 theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3′ ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme BsaI, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins.

DOI： 10.21769/BioProtoc.4125

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Language：English   Publishing type：Research paper (scientific journal)  

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.

DOI： 10.1039/d1ob00995h

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

We synthesized Fmoc-propargyl glycine derivatives bearing different silyl protecting groups that can be readily introduced by using a standard solid-phase peptide coupling procedures. Taking advantage of the orthogonality between the different silyl protecting groups, chemoselective incorporation of functional molecules into a 19-mer peptide through click reactions was demonstrated.

DOI： 10.1016/j.tetlet.2021.153093

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

<p>Chemical protein synthesis assisted by an organoruthenium catalyst streamlined the production of heterochromatin factors bearing various patterns of epigenetic modifications, and their biological significance was elucidated.</p>

DOI： 10.1039/d1sc00731a

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Language：English   Publishing type：Research paper (scientific journal)  

We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently attached to the cDNA. As a proof-of-concept, we demonstrated a rapid model selection of an anti-EGFR1 monobody in a solution containing ribonuclease.

DOI： 10.1039/d0cc07541h

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.26434/chemrxiv.14721477.v1

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nucleic Acids Research  

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

DOI： 10.1093/nar/gkaa949

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry  

We report an effective fluorescence in situ hybridization strategy, named l-DNA tagged FISH (LT-FISH), for highly sensitive RNA detection in fixed cultured cells. LT-FISH includes two-step hybridization processes with a l-d chimera oligonucleotide probe and a fluorescence-labeled PCR product tethering a l-DNA tag. The degree of fluorescence enhancement, depending on the length of PCR products, was up to 14-fold when the 606 bp product was used. Endogenous mRNA and miRNA in cancer cells were visualized by utilizing this l-DNA-mediated signal amplification technique.

DOI： 10.1039/d0ob01635g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Current Opinion in Chemical Biology  

Histone post-translational modifications play significant roles in gene regulation processes. Among many approaches, chemical protein synthesis has been a successful and promising method for the preparation of homogeneous products of site-specifically modified histones for elucidation of their biological significance. In this short review, we describe the recent advances in synthetic toolbox for histone proteins such as thioester precursors, chemical ubiquitination, and one-pot peptide ligation.

DOI： 10.1016/j.cbpa.2020.04.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Science Advances  

To combat severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

DOI： 10.1126/sciadv.abd3916

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Organic Letters  

We report an Fmoc-compatible and external-thiol-free method of peptide C-terminus thioesterification with cysteinylprolyl imide. The newly synthesized structure, i.e., cysteinylprolyl-thiazolidinone, provided high conversion and sequence-independent fast kinetics (90 min) in the diketopiperazine thioester formation under relatively mild conditions: pH 6.0, 37 °C. Employing this thioesterification method, we synthesized histone H3.2 bearing K56 acetylation.

DOI： 10.1021/acs.orglett.0c01450

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DOI： 10.5059/yukigoseikyokaishi.78.130

Language：English   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本薬学会  

タンパク質化学合成法の発展により、多様な翻訳後修飾を持つタンパク質の作製が可能なってきた。本稿では、タンパク質化学合成法について解説するとともに、化学合成タンパク質を用いた翻訳後修飾研究について紹介する。特に、エピジェネティクス研究で中心的役割を果たすヒストンタンパク質の翻訳後修飾研究、また本特集のテーマであるユビキチン鎖やユビキチン化タンパク質を化学的に合成し、応用した研究例について紹介する。

DOI： 10.14894/faruawpsj.56.1_46

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：The Society of Synthetic Organic Chemistry, Japan  

<p>Chemical protein synthesis (CPS) that consists of solid-phase peptide synthesis and peptide ligation generates not only naturally-occurring proteins with/without posttranslational modifications (PTMs), but also a variety of artificial proteins including unnatural amino-acids such as ᴅ-amino acids and fluorophore-labeled amino acids. This unique property offers new analytical methods for protein structure and interaction in terms of PTMs. In fact, we have chemically synthesized histone proteins with PTMs, which play an important role in regulation of gene expression in eukaryotic cells, and analyzed the effects of PTMs such as methylation, acetylation, and phosphorylation. However, current CPS is still in developing process and includes several issues to be solved such as difficult handling in hydrophobic protein synthesis and time-consuming multistep process for large protein synthesis. We have approached these issues by creating new strategies for peptide ligation. One-pot ligation of five peptide segments was demonstrated for the first time by utilizing multifunctionality of thiophenol compound in deprotection of allyloxycarbonyl group by palladium complex. New thioester precursors for native chemical ligation, which have potential to offer a novel two-way one-pot ligation, have also been developed recently. Furthermore, we have been developing a new strategy for simultaneous ligation of multiple peptides on DNA scaffold, which can connect peptides in highly diluted condition. This article also describes the background and current situation of CPS with our future perspectives.</p>

DOI： 10.5059/yukigoseikyokaishi.78.130

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DOI： 10.26434/chemrxiv.13340582.v1

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DOI： 10.26434/chemrxiv.12154722.v1

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Organic Letters  

We report selective removals of N-terminal and internal Cys protecting groups using different palladium complexes to facilitate the efficient chemical protein synthesis. Utilizing the orthogonal deprotection pairs, we accomplished chemical synthesis of histone H3 containing trimethylated Lys through the combination of Pd(0)-mediated Alloc deprotection for one-pot multiple peptide ligation and Pd(II)Cl2-mediated Acm deprotection to recover native Cys residues after desulfurization.

DOI： 10.1021/acs.orglett.9b03152

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Chemical Science  

Native chemical ligation (NCL) between the C-terminal peptide thioester and the N-terminal cysteinyl-peptide revolutionized the field of chemical protein synthesis. The difficulty of direct synthesis of the peptide thioester in the Fmoc method has prompted the development of crypto-thioesters that can be efficiently converted into thioesters. Cysteinylprolyl ester (CPE), which is an N-S acyl shift-driven crypto-thioester that relies on an intramolecular O-N acyl shift to displace the amide-thioester equilibrium, enabled trans-thioesterification and subsequent NCL in one pot. However, the utility of CPE is limited because of the moderate thioesterification rates and the synthetic complexity introduced by the ester group. Herein, we develop a new crypto-thioester, cysteinylprolyl imide (CPI), which replaces the alcohol leaving group of CPE with other leaving groups such as benzimidazolidinone, oxazolidinone, and pyrrolidinone. CPI peptides were efficiently synthesized by using standard Fmoc solid-phase peptide synthesis (SPPS) and subsequent on-resin imide formation. Screening of the several imide structures indicated that methyloxazolidinone-t-leucine (MeOxd-Tle) showed faster conversion into thioester and higher stability against hydrolysis under NCL conditions. Finally, by using CPMeOxd-Tle peptides, we demonstrated the chemical synthesis of affibody via N-to-C sequential, three-segment ligation and histone H2A.Z via convergent four-segment ligation. This facile and straightforward method is expected to be broadly applicable to chemical protein synthesis.

DOI： 10.1039/c9sc00646j

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DOI： 10.1021/acs.biomac.8b01655

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DOI： 10.1002/anie.201814304

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DOI： 10.1002/anie.201809765

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DOI： 10.1002/tcr.201800040

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DOI： 10.1039/c8cc01965g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

<p>Intracellular delivery of oligonucleotides is essential not only for antisense- and RNAi-therapeutics, but also for live-cell RNA imaging with fluorescent nucleic acid probes. Herein, we developed oligonucleotide–peptide conjugates synthesized via Cu-catalyzed alkyne-azide cycloaddition between an exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probe and linear or cyclic cell-penetrating peptides (CPPs). When lipofected to living HeLa cells, the CPP-conjugated ECHO probe showed enhanced cellular uptake efficiency compared with that of the unconjugated ECHO probe.</p>

DOI： 10.1246/cl.170813

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/acs.biochem.7b00504

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DOI： 10.1039/c7cc02612a

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DOI： 10.1038/srep41007

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Language：English   Publishing type：Research paper (scientific journal)  

5-Hydroxymethylcytosine (hmC) is an essential intermediate in the active DNA demethylation pathway. Here we report a new base-resolution method for measuring hmC by combining peroxotungstate-mediated oxidation and sequencing analysis. We reveal that an oxidized product of hmC, trihydroxylated thymine (thT), tolerated the incorporation of dATP as a substrate in the process of DNA polymerase elongation. By comparing the results of Sanger sequencing before and after the oxidation, we observed that hmC sites on single-stranded DNAs could be discriminated from unmethylated cytosines. We found that a thermal cycle condition during peroxotungstate treatment enhanced the oxidation reaction of hmC in double-stranded DNA. Furthermore, Illumina sequencing analysis of hmC-containing synthetic genome fragments enabled us to identify simultaneously the positions of hmC in base resolution. This bisulfite-free simple hmC detection technique could facilitate the acquisition of epigenomic information.

DOI： 10.1021/jacs.6b06428

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DOI： 10.1039/c5cc10555b

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

N-6-methyladenosine (m(6)A) is a prevalent modification of RNAs. m(6)A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation, We applied diazirine photocrosslinking to the event of m(6)A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m(6)A on the RNA successfully recruited activated FTO complexes with an m(6)A preference. The process of recognition of m(6)A via FTO using diazirine photocrosslinking was controlled by the alpha-ketoglutarate (alpha-KG) cosubstrate and the Peal). cofactor, which are involved in m(6)A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m(6)A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m(6)A recognition and provides new insights into the dynamic FTO oxidative demethylation process.

DOI： 10.1021/cb5010096

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DOI： 10.1002/asia.201500172

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DOI： 10.1021/acs.bioconjchem.5b00090

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Mass Spectrometry Society of Japan  

Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach.Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments.This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides.

DOI： 10.5702/massspectrometry.A0039

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

DOI： 10.1038/nchembio.1549

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CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-1-62703-755-6_3

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/tcr.201200026

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1039/c2ob26707a

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2012.10.072

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1098/rstb.2011.0137

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.chembiol.2010.04.005

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/chem.200800936

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiotec.2008.03.011

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrn355

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1021/ja071298x

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbic.200600477

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/nrm047

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Language：English   Publishing type：Research paper (scientific journal)  

We here show the first identified ligand 2,7-diamino-1,8-naphthyridine (DANP) that strongly and specifically binds to the single cytosine and thymine bulges with exclusively 1:1 stoichiometry.

DOI： 10.1016/j.bmc.2005.04.035

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DNA trinucleotide repeats, particularly CXG, are common within the human genome. However, expansion of trinucleotide repeats is associated with a number of disorders, including Huntington disease, spinobulbar muscular atrophy and spinocerebellar ataxia. In these cases, the repeat length is known to correlate with decreased age of onset and disease severity. Repeat expansion of (CAG)n, (CTG)n and (CGG)n trinucleotides may be related to the increased stability of alternative DNA hairpin structures consisting of CXG-CXG triads with X-X mismatches. Small-molecule ligands that selectively bound to CAG repeats could provide an important probe for determining repeat length and an important tool for investigating the in vivo repeat extension mechanism. Here we report that napthyridine-azaquinolone (NA, 1) is a ligand for CAG repeats and can be used as a diagnostic tool for determining repeat length. We show by NMR spectroscopy that binding of NA to CAG repeats induces the extrusion of a cytidine nucleotide from the DNA helix.

DOI： 10.1038/nchembio708

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/nass/49.1.261

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Language：English   Publishing type：Research paper (scientific journal)  

NMR structure of the first identified ligand, naphthyridine-azaquinolone (NA), complexed with the CAG-CAG triad is reported. The determined structure revealed the invasive ligands binding to the A-A mismatch and flanking G-C base pairs, causing the widowed cytosines to flip out from pi-stack. Hydrogen-bond pairs between NA and DNA, naphthyridine-guanine and azaquinolone-adenine, are well stacked in the right-handed DNA helix, showing structural mimicry of Watson-Crick base pairing. This is the first observation that the small molecular ligand induced the base flipping of the nucleotide base in the Watson-Crick base pair.

DOI： 10.1093/nass/49.1.49

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Language：English   Publishing type：Research paper (scientific journal)  

We have discovered a new molecule naphthyridine-azaquinolone hybrid (Npt-Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt-Azq, the melting temperature (T(m)) of 5'-d(CTA ACG GAA TG)-3'/3'-d(GAT TGA CTT AC)-5' containing a single G-A mismatch increased by 15.4 degrees C, whereas fully matched duplex increased its T(m) only by 2.2 degrees C. Npt-Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt-Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was approximately 6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 microM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt-Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.

DOI： 10.1093/nar/gkh171

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Language：English   Publishing type：Research paper (scientific journal)  

Discrimination of mismatched base pairs having various flanking sequence from normal Watson-Crick base pairs were examined using SPR sensor surface where mismatch binding ligand (MBL) were immobilized. CC, CT, and TT mismatched base pair are distinguishable from wild type DNA by means of diaminonaphthyndine dimer (damND) immobilized sensor surface. By comparing the component ratio of responses obtained by MLB immobilized sensors, distinct differences could be observed between mismatched base pairs having almost the same total SPR response.

DOI： 10.1093/nass/48.1.129

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Language：English  

DOI： 10.1002/asia.201500172

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Language：English  

DOI： 10.1021/acs.bioconjchem.5b00090

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Language：English  

DOI： 10.5702/massspectrometry.A0039

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Language：English  

DOI： 10.1038/nchembio.1549

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Language：English  

DOI： 10.1007/978-1-62703-755-6_3

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Language：English  

DOI： 10.1002/tcr.201200026

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Language：English  

DOI： 10.1039/c2ob26707a

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Language：English  

DOI： 10.1016/j.bbrc.2012.10.072

PubMed

Language：English  

DOI： 10.1098/rstb.2011.0137

PubMed

Language：Japanese  

PubMed

Language：English  

DOI： 10.1016/j.chembiol.2010.04.005

PubMed

Language：English  

DOI： 10.1002/chem.200800936

PubMed

Language：English  

DOI： 10.1016/j.jbiotec.2008.03.011

PubMed

Language：English  

DOI： 10.1093/nass/nrn355

PubMed

Language：English  

DOI： 10.1021/ja071298x

PubMed

Language：English  

DOI： 10.1002/cbic.200600477

PubMed

Language：English  

DOI： 10.1093/nass/nrm047

PubMed

Language：English  

DOI： 10.1016/j.bmc.2005.04.035

PubMed

Language：English  

DOI： 10.1038/nchembio708

PubMed

Language：English  

DOI： 10.1093/nass/49.1.261

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Language：English  

DOI： 10.1093/nass/49.1.49

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Language：English  

DOI： 10.1093/nass/48.1.129

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Language：English  

DOI： 10.1093/nar/gkh171

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