研究者詳細 - 木村　康明

論文 - 木村　康明

分割表示 54 件中 21 - 40 件目／全件表示 >>

A 2'-modified uridine analog, 2'-O-(methylthiomethoxy)methyl uridine, for siRNA applications. 査読有り Open Access

Lyu F, An S, Kobayashi Y, Nomura K, Baba R, Abe N, Hiraoka H, Hashiya F, Shu Z, Ui-Tei K, Kimura Y, Abe H

Bioorganic & medicinal chemistry letters 74 巻頁： 128939 - 128939 2022年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioorganic and Medicinal Chemistry Letters

The medicinal applications of siRNAs have been intensively examined but are still hindered by their low molecular stability under biological conditions and off-target effects, etc. The introduction of chemical modifications to the nucleoside is a promising strategy for solving these limitations. Herein, we describe the development of a new uridine analog, U*, that has a (methylthiomethoxy)methoxy group at the 2′ position. The phosphoramidite reagent corresponding to U* was easily synthesized and the RNA oligonucleotides containing U* were stably prepared using a standard protocol for oligonucleotide synthesis. The introduction of U* into the siRNA resulted in positive or negative effects on the targeted gene silencing in a position-dependent manner, and the positive effects were attributed to the improved stability under biological conditions. The thermodynamic analysis of the U*-modified RNAs revealed a slight destabilization of the dsRNA, based depending on which U was strategically utilized to restrain the off-target effects of the siRNA. This study describes a rare example of nucleoside analogs with a large substitution at the 2′-position in the context of an siRNA application and is informative for the development of other analogs to further improve the molecular properties of siRNAs for medicinal applications.

DOI： 10.1016/j.bmcl.2022.128939

Cap analogs with a hydrophobic photocleavable tag enable facile purification of fully capped mRNA with various cap structures.

Masahito Inagaki, Naoko Abe, Zhenmin Li, Yuko Nakashima, Susit Acharyya, Kazuya Ogawa, Daisuke Kawaguchi, Haruka Hiraoka, Ayaka Banno, Zheyu Meng, Mizuki Tada, Tatsuma Ishida, Pingxue Lyu, Kengo Kokubo, Hirotaka Murase, Fumitaka Hashiya, Yasuaki Kimura, Satoshi Uchida, Hiroshi Abe

2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：American Chemical Society (ACS)

Removing immunogenic uncapped mRNA from in vitro transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide a maximum capping efficiency of around 80-90% for widely used Cap-0- and Cap-1-type mRNAs. However, uncapped and capped mRNA possesses almost identical physicochemical properties, posing challenges to their physical separation. Herein, we developed hydrophobic photocaged tag-modified cap analogs, which separated capped mRNA from uncapped mRNA by reversed-phase HPLC. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provided 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. The Cap-2-type mRNA showed up to 3 to 4-fold higher translational activity in cultured cells and animals than mRNA prepared by the standard capping method. Notably, the purification process simultaneously removed immunogenic double-stranded mRNA, another major contaminant of in vitro transcribed mRNA, drastically reducing mRNA immunogenicity in cultured cells.

DOI： 10.26434/chemrxiv-2022-vfjt6

Development of Fluorophosphoramidate as a Biocompatibly Transformable Functional Group and its Application as a Phosphate Prodrug for Nucleoside Analogs. 査読有り国際誌

Yoshida Y, Zheng T, Tanabe W, Tomoike F, Hashiya F, Suzuki T, Hirota S, Saiki Y, Horii A, Hirayama A, Soga T, Kimura Y, Abe H

ChemMedChem 17 巻 ( 17 ) 頁： e202200188 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemMedChem

Synthetic phosphate-derived functional groups are important for controlling the function of bioactive molecules in vivo. Herein we describe the development of a new type of biocompatible phosphate analog, a fluorophosphoramidate (FPA) functional group that has characteristic P−F and P−N bonds. We found that FPA with a primary amino group was relatively unstable in aqueous solution and was converted to a monophosphate, while FPA with a secondary amino group was stable. Furthermore, by improving the molecular design of FPA, we developed a reaction in which a secondary amino group is converted to a primary amino group in the intracellular environment and clarified that the FPA group functions as a phosphate prodrug of nucleoside. Various FPA-gemcitabine derivatives were synthesized and their toxicity to cancer cells were evaluated. One of the FPA-gemcitabine derivatives showed superior toxicity compared with gemcitabine and its ProTide prodrug, which methodology is widely used in various nucleoside analogs, including anti-cancer and anti-virus drugs.

DOI： 10.1002/cmdc.202200188

Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction. 査読有り国際誌

Abe N, Imaeda A, Inagaki M, Li Z, Kawaguchi D, Onda K, Nakashima Y, Uchida S, Hashiya F, Kimura Y, Abe H

ACS chemical biology 17 巻 ( 6 ) 頁： 1308 - 1314 2022年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Chemical Biology

Site-specific chemical modification of mRNA can improve its translational efficiency and stability. For this purpose, it is desirable to develop a complete chemical synthesis method for chemically modified mRNA. The key is a chemical reaction that introduces a cap structure into the chemically synthesized RNA. In this study, we developed a fast and quantitative chemical capping reaction between 5′-phosphorylated RNA and N7-methylated GDP imidazolide in the presence of 1-methylimidazole in the organic solvent dimethyl sulfoxide. It enabled quantitative preparation of capping RNA within 3 h. We prepared chemically modified 107-nucleotide mRNAs, including N6-methyladenosine, insertion of non-nucleotide linkers, and 2′-O-methylated nucleotides at the 5′ end and evaluated their effects on translational activity in cultured HeLa cells. The results showed that mRNAs with non-nucleotide linkers in the untranslated regions were sufficiently tolerant to translation and that mRNAs with the Cap_2 structure had higher translational activity than those with the Cap_0 structure.

DOI： 10.1021/acschembio.1c00996

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization. 査読有り国際誌

Hiraoka H, Shu Z, Tri Le B, Masuda K, Nakamoto K, Fangjie L, Abe N, Hashiya F, Kimura Y, Shimizu Y, Veedu RN, Abe H

Chembiochem : a European journal of chemical biology 22 巻 ( 24 ) 頁： 3437 - 3442 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemBioChem

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.

DOI： 10.1002/cbic.202100413

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Yamaoka K, Oikawa R, Abe N, Nakamoto K, Tomoike F, Hashiya F, Kimura Y, Abe H

Chembiochem : a European journal of chemical biology 22 巻 ( 23 ) 頁： 3273 - 3276 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemBioChem

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.

DOI： 10.1002/cbic.202100312

Structure, Synthesis and Inhibition Mechanism of Nucleoside Analogues as HIV-1 Reverse Transcriptase Inhibitors (NRTIs). 査読有り国際誌

Yoshida Y, Honma M, Kimura Y, Abe H

ChemMedChem 16 巻 ( 5 ) 頁： 743 - 766 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemMedChem

Acquired immunodeficiency syndrome (AIDS) is caused by infection with the human immunodeficiency virus (HIV). Although treatments against HIV infection are available, AIDS remains a serious disease that causes many deaths annually. Although a variety of anti-HIV drugs have been synthesized and marketed to treat HIV-infected patients, nucleoside analogue reverse transcriptase inhibitors (NRTIs), which mimic nucleosides, are used extensively and remain a subject of interest to medicinal chemists. However, HIV has acquired drug resistance against NRTIs, and thus the struggle to find novel therapies continues. In this review, we trace the trajectory of NRTIs, focusing on the synthesis, mechanisms of action and applications of NRTIs that have been developed.

DOI： 10.1002/cmdc.202000695

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cmdc.202000695

Phosphorothioate Modification of mRNA Accelerates the Rate of Translation Initiation to Provide More Efficient Protein Synthesis. 査読有り国際誌 Open Access

Kawaguchi D, Kodama A, Abe N, Takebuchi K, Hashiya F, Tomoike F, Nakamoto K, Kimura Y, Shimizu Y, Abe H

Angewandte Chemie (International ed. in English) 59 巻 ( 40 ) 頁： 17403 - 17407 2020年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Angewandte Chemie - International Edition

Messenger RNAs (mRNAs) with phosphorothioate modification (PS-mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell-free translation system. Protein synthesis from PS-mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22-fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′-end to the initiation codon. Single-turnover analysis of PS-mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non-natural mRNA.

DOI： 10.1002/anie.202007111

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/anie.202007111

Free-Energy Calculation of Ribonucleic Inosines and Its Application to Nearest-Neighbor Parameters 査読有り国際誌

Sakuraba Shun, Iwakiri Junichi, Hamada Michiaki, Kameda Tomoshi, Tsuji Genichiro, Kimura Yasuaki, Abe Hiroshi, Asai Kiyoshi

JOURNAL OF CHEMICAL THEORY AND COMPUTATION 16 巻 ( 9 ) 頁： 5923 - 5935 2020年9月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Chemical Theory and Computation

Can current simulations quantitatively predict the stability of ribonucleic acids (RNAs)? In this research, we apply a free-energy perturbation simulation of RNAs containing inosine, a modified ribonucleic base, to the derivation of RNA nearest-neighbor parameters. A parameter set derived solely from 30 simulations was used to predict the free-energy difference of the RNA duplex with a mean unbiased error of 0.70 kcal/mol, which is a level of accuracy comparable to that obtained with parameters derived from 25 experiments. We further show that the error can be lowered to 0.60 kcal/mol by combining the simulation-derived free-energy differences with experimentally measured differences. This protocol can be used as a versatile method for deriving nearest-neighbor parameters of RNAs with various modified bases.

DOI： 10.1021/acs.jctc.0c00270

Chemically synthesized circular RNAs with phosphoramidate linkages enable rolling circle translation. 査読有り国際誌

Nakamoto K, Abe N, Tsuji G, Kimura Y, Tomoike F, Shimizu Y, Abe H

Chemical communications (Cambridge, England) 56 巻 ( 46 ) 頁： 6217 - 6220 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Chemical Communications

Circular RNA without a stop codon enables rolling circle translation. To produce circular RNAs, we carried out one-pot chemical synthesis of circular RNA from RNA fragments with the use of an EDC/HOBt-based chemical ligation reaction. The synthesized circular RNAs acted as translation templates, despite the presence of unnatural phosphoramidate linkages.

DOI： 10.1039/d0cc02140g

Quantification of native mRNA dynamics in living neurons using fluorescence correlation spectroscopy and reduction-triggered fluorescent probes. 査読有り国際誌 Open Access

Fujita H, Oikawa R, Hayakawa M, Tomoike F, Kimura Y, Okuno H, Hatashita Y, Fiallos Oliveros C, Bito H, Ohshima T, Tsuneda S, Abe H, Inoue T

The Journal of biological chemistry 295 巻 ( 23 ) 頁： 7923 - 7940 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

RNA localization in subcellular compartments is essential for spatial and temporal regulation of protein expression in neurons. Several techniques have been developed to visualize mRNAs inside cells, but the study of the behavior of endogenous and nonengineered mRNAs in living neurons has just started. In this study, we combined reduction-triggered fluorescent (RETF) probes and fluorescence correlation spectroscopy (FCS) to investigate the diffusion properties of activity-regulated cytoskeleton-associated protein (Arc) and IP3 receptor isoform 1 (Ip3r1) mRNAs. This approach enabled us to discriminate between RNA-bound and unbound fluorescent probes and to quantify mRNA diffusion parameters and concentrations in living rat primary hippocampal neurons. Specifically, we detected the induction of Arc mRNA production after neuronal activation in real time. Results from computer simulations with mRNA diffusion coefficients obtained in these analyses supported the idea that free diffusion is incapable of transporting mRNA of sizes close to those of Arc or Ip3r1 to distal dendrites. In conclusion, the combined RETF–FCS approach reported here enables analyses of the dynamics of endogenous, unmodified mRNAs in living neurons, affording a glimpse into the intracellular dynamics of RNA in live cells.

DOI： 10.1074/jbc.RA119.010921

A robust model for quantitative prediction of the silencing efficacy of wild-type and A-to-I edited miRNAs. 査読有り国際誌 Open Access

Tian S, Terai G, Kobayashi Y, Kimura Y, Abe H, Asai K, Ui-Tei K

RNA biology 17 巻 ( 2 ) 頁： 264 - 280 2020年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：RNA Biology

MicroRNAs (miRNAs) are small non-coding RNAs that play essential roles in the regulation of gene function by a mechanism known as RNA silencing. In a previous study, we revealed that miRNA-mediated silencing efficacy is correlated with the combinatorial thermodynamic properties of the miRNA seed–target mRNA duplex and the 5´-terminus of the miRNA duplex, which can be predicted using ‘miScore’. In this study, a robust refined-miScore was developed by integrating the thermodynamic properties of various miRNA secondary structures and the latest thermodynamic parameters of wobble base-pairing, including newly established parameters for I:C base pairing. Through repeated random sampling and machine learning, refined-miScore models calculated with either melting temperature (Tm) or free energy change (ΔG) values were successfully built and validated in both wild-type and adenosine-to-inosine edited miRNAs. In addition to the previously reported contribution of the seed–target duplex and 5´-terminus region, the refined-miScore suggests that the central and 3´-terminus regions of the miRNA duplex also play a role in the thermodynamic regulation of miRNA-mediated silencing efficacy.

DOI： 10.1080/15476286.2019.1678364

Intracellular build-up RNAi with single-strand circular RNAs as siRNA precursors. 査読有り国際誌

Kimura Y, Shu Z, Ito M, Abe N, Nakamoto K, Tomoike F, Shuto S, Ito Y, Abe H

Chemical communications (Cambridge, England) 56 巻 ( 3 ) 頁： 466 - 469 2020年1月

担当区分：筆頭著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Royal Society of Chemistry ({RSC})

We herein report a new approach for RNA interference, so-called "build-up RNAi" approach, where single-strand circular RNAs with a photocleavable unit or disulfide moiety were used as siRNA precursors. The advantages of using these circular RNA formats for RNAi were presented in aspects of immunogenicity and cellular uptake.

DOI： 10.1039/c9cc04872c

SYNTHESIS AND BIOLOGICAL EVALUATION OF NMDI14 DERIVATIVES AS ANTI-MESOTHELIOMA AGENTS 査読有り国際誌

Hong Nhung Nguyen, Suzuki Koya, Kimura Yasuaki, Hirokawa Takatsugu, Murakami-Tonami Yuko, Abe Hiroshi

Heterocycles 100 巻 ( 2 ) 頁： 253 - 266 2020年1月

Translational control by secondary-structure formation in mRNA in a eukaryotic system. 査読有り国際誌 Open Access

Kawaguchi D, Shimizu S, Abe N, Hashiya F, Tomoike F, Kimura Y, Abe H

Nucleosides, nucleotides & nucleic acids 39 巻 ( 1-3 ) 頁： 195 - 203 2020年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nucleosides, Nucleotides and Nucleic Acids

Eukaryotic mRNA has a cap structure at the 5′ end and a poly(A) tail at the 3′ end. The cap and poly(A) tail form a complex with multiple translation factors, and mRNA forms a circularized structure called a closed-loop model. This circularized structure reportedly not only stabilizes mRNA but also promotes ribosome recycling during translation, which improves translation efficiency. We designed an artificial mRNA that forms a circularized structure without a cap structure and poly(A) tail and found that its translational efficiency was improved compared with that of a sequence without the circularized structure in a eukaryotic translation system.

DOI： 10.1080/15257770.2019.1671593

Intracellular delivery of Antisense DNA and siRNA with amino groups masked with disulfide units 査読有り国際誌

Zhaoma Shu, Azumi Ota, Yukiya Takayama, Yuri Katsurada, Kosuke Kusamori, Naoko Abe, Kosuke Nakamoto, Fumiaki Tomoike, Seiichi Tada, Yoshihiro Ito, Makiya Nishikawa, Yasuaki Kimura, Hiroshi Abe

Chemical and Pharmaceutical Bulletins 68 巻 ( 2 ) 頁： 129 - 132 2019年12月

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA 査読有り国際誌

Zhaoma Shu, Iku Tanaka, Azumi Ota, Daichi Fushihara, Naoko Abe, Saki Kawaguchi, Kosuke Nakamoto, Fumiaki Tomoike, Seiichi Tada, Yoshihiro Ito, Yasuaki Kimura, Hiroshi Abe

Angewandte Chemie 131 巻 ( 20 ) 頁： 6683 2019年5月

A Covalent Inhibitor for Glutathione S-Transferase Pi (GSTP(1-1) ) in Human Cells. 査読有り国際誌

Shishido Y, Tomoike F, Kuwata K, Fujikawa H, Sekido Y, Murakami-Tonami Y, Kameda T, Abe N, Kimura Y, Shuto S, Abe H

Chembiochem : a European journal of chemical biology 20 巻 ( 7 ) 頁： 900 - 905 2019年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ChemBioChem

Glutathione S-transferase π (GSTP 1-1 ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP 1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP 1-1 both in vitro and in cellulo with a long duration of action.

DOI： 10.1002/cbic.201800671

その他リンク： http://orcid.org/0000-0002-3609-602X

膜透過性を有するGST共有結合性阻害剤の開発

友池史明, 宍戸裕子, 藤川遥加, 木村康明, 桑田啓子, 村上優子, 福井健二, 関戸好孝, 矢野貴人, 亀田倫史, 周東智, 阿部洋

日本薬学会年会要旨集 139年会巻 ( 2 ) 頁： 80 - 80 2019年3月

N(6)-methyl adenosine in siRNA evades immune response without reducing RNAi activity. 査読有り国際誌

Imaeda A, Tomoike F, Hayakawa M, Nakamoto K, Kimura Y, Abe N, Abe H

Nucleosides, nucleotides & nucleic acids 38 巻 ( 12 ) 頁： 972 - 979 2019年

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