Language：English  

DOI： 10.1021/acsomega.3c08516

Web of Science

Scopus

PubMed

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

DOI： 10.1039/d2cc06189

Web of Science

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

Controlling PCR fidelity is an important issue for molecular biology and high-fidelity PCR is essential for gene cloning. In general, fidelity control is achieved by protein engineering of polymerases. In contrast, only a few studies have reported controlling fidelity using chemically modified nucleotide substrates. In this report, we synthesized nucleotide substrates possessing a modification on Pγ and evaluated the effect of this modification on PCR fidelity. One of the substrates, nucleotide tetraphosphate, caused a modest decrease in Taq DNA polymerase activity and the effect on PCR fidelity was dependent on the type of mutation. The use of deoxyadenosine tetraphosphate enhanced the A:T→G:C mutation dramatically, which is common when using Taq polymerase. Conversely, deoxyguanosine tetraphosphate (dG4P) suppressed this mutation but increased the G:C→A:T mutation during PCR. Using an excess amount of dG4P suppressed both mutations successfully and total fidelity was improved.

DOI： 10.1002/cbic.202200572

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Scopus

PubMed

Language：English   Publisher：Nature Communications  

Starting with the clinical application of two vaccines in 2020, mRNA therapeutics are currently being investigated for a variety of applications. Removing immunogenic uncapped mRNA from transcribed mRNA is critical in mRNA research and clinical applications. Commonly used capping methods provide 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. In this work, we develop hydrophobic photocaged tag-modified cap analogs, which separate capped mRNA from uncapped mRNA by reversed-phase high-performance liquid chromatography. Subsequent photo-irradiation recovers footprint-free native capped mRNA. This approach provides 100% capping efficiency even in Cap-2-type mRNA with versatility applicable to 650 nt and 4,247 nt mRNA. We find that the Cap-2-type mRNA shows up to 3- to 4-fold higher translation activity in cultured cells and animals than the Cap-1-type mRNA prepared by the standard capping method.

DOI： 10.1038/s41467-023-38244-8

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PubMed

Language：English   Publisher：Japanese Society for Medical and Biological Engineering  

<p>Fine motor dysfunction and cognitive impairments commonly develop after stroke, which greatly impact the daily lives of patients. In current occupational therapy, hand dexterity and cognitive functions are evaluated individually (e.g., by manipulation of small objects with fingers, or a paper-and-pencil test), which is insufficient for therapists to grasp the total ability of combined dexterity and cognition in everyday situations. Additionally, the traditional methods require a tester to measure the completion time manually and tend to be monotonous for patients. These problems would be solved using technology. This study aimed to develop a new electric pegboard (e-Peg) prototype and to investigate preliminary utility in healthy adults. The system judges the peg insertion accuracy based on magnetism and records the time course and scores, which are linked to human object manipulation ability. The e-Peg executes three types of tasks: a basic color matching task (BT), a color comparison task using a pattern sheet (CT), and a visual memory task (MT), with one/two-color sample patterns. Six older and nine younger healthy adults performed the e-Peg tasks, functional tests, and responded to questionnaires. As a result, the number of correct answers in a bicolor symmetrical MT were significantly greater in the younger group than in the older group. The older group required a significantly longer completion time for BT and CT than the younger group. Significant correlations were found between one-color BT/CT and dexterity tests, between bicolor BT/CT and dexterity/cognitive tests, and between a bicolor MT and a cognitive test. Questionnaire results revealed that participants regarded BT/CT as easy/interesting tasks, whereas MT was considered a difficult/challenging task. In conclusion, our e-Peg is potentially a useful rehabilitation device that facilitates many tasks related to hand manipulation and attention/executive functions, and a valuable tool for personalized therapy.</p>

DOI： 10.14326/abe.12.81

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Scopus

CiNii Research

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

DOI： 10.1016/j.bmcl.2022.128939

Web of Science

Scopus

PubMed

Publisher：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

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

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

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Scopus

PubMed

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

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

Web of Science

Scopus

PubMed

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

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

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Scopus

PubMed

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

DOI： 10.1002/cbic.202100312

Web of Science

Scopus

PubMed

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

DOI： 10.1002/cbic.202100004

Web of Science

Scopus

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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

The nucleosome is one of the most fundamental units involved in gene expression and consequent cell development, differentiation, and expression of cell functions. We report here a method to place reconstituted nucleosomes into a DNA origami frame for direct observation using high-speed atomic-force microscopy (HS-AFM). By using this method, multiple nucleosomes can be incorporated into a DNA origami frame and real-time movement of nucleosomes can be visualized. The arrangement and conformation of nucleosomes and the distance between two nucleosomes can be designed and controlled. In addition, four nucleosomes can be placed in a DNA frame. Multiple nucleosomes were well accessible in each conformation. Dynamic movement of the individual nucleosomes were precisely monitored in the DNA frame, and their assembly and interaction were directly observed. Neither mica surface modification nor chemical fixation of nucleosomes is used in this method, meaning that the DNA frame not only holds nucleosomes, but also retains their natural state. This method offers a promising platform for investigating nucleosome interactions and for studying chromatin structure.

DOI： 10.1002/chem.202003071

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PubMed

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

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

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PubMed

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

We developed an epigenetically active, cooperative DNA binding transcription factor platform assisted by cucurbit[7]uril (CB7) host-guest modules. This new type of molecule termed ePIP-HoGu not only mimics the operation of transcription factors as a pair but also recruits the epigenetic modifier to a particular DNA locus.

DOI： 10.1039/c9cc09608f

PubMed

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

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

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Scopus

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Electron transfer through π-stacked arrays of double-stranded DNA contributes to the redox chemistry of bases, including guanine oxidation and thymine-thymine dimer repair by photolyase. 5-Bromouracil is an attractive photoreactive thymine analogue that can be used to investigate electron transfer in DNA, and is a useful probe for protein-DNA interaction analysis. In the present study using BrU we found that UV irradiation facilitated electron injection from mitochondrial transcription factor A into DNA. We also observed that this electron injection could lead to repair of a thymine-thymine dimer.

DOI： 10.1016/j.bmc.2018.11.044

PubMed

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

A distance- and orientation-factor-dependent FRET system is a useful and attractive approach to the investigation of the conformational dynamics of nucleosomes. In this study, the application of the highly emissive nucleobase th dG-tC FRET pair to 601 nucleosomes is reported. It was found that the th dG-tC FRET pair was successfully incorporated to 145 bp 601 sequences, and different FRET efficiencies were obtained for the designated donor and acceptor positions in the nucleosome.

DOI： 10.1002/chem.201803382

PubMed

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

Despite evidence that histone H3 and H4 proteins may act as the precursor for orientating the DNA sequence to form nucleosome structures, there is no direct evidence of the formed compact structure. Here, it is demonstrated that a histone H3-H4 octasome could be constructed without the involvement of histone H2A-H2B under in vitro reconstitution conditions. Atomic force microscopy was used to obtain the first direct observation of the octasome structure, which exhibited a similar core-protein size as that of a nucleosome but with a shorter core histone-binding DNA region. The octasome also displayed a one-step histone-dissociation pattern under heat treatment, distinct micrococcal nuclease and peplomycin accessibility, which suggests a different wrapping pattern to that in nucleosomes.

DOI： 10.1002/chem.201804010

PubMed

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

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole-imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner.

DOI： 10.1021/jacs.8b01518

PubMed

Language：English  

Authorship：Lead author   Language：English  

Language：English  

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

Event date： 2024.9

Language：English   Presentation type：Poster presentation  

Country：Japan  

Event date： 2024.9

Language：English   Presentation type：Poster presentation  

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Event date： 2024.9

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Venue：Suzhou, China   Country：China  

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Venue：星薬科大学   Country：Japan  

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Venue：パシフィコ横浜　会議センター・展示ホールAB   Country：Japan  

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Venue：パシフィコ横浜　会議センター・展示ホールAB   Country：Japan  

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Venue：パシフィコ横浜　会議センター・展示ホールAB   Country：Japan  

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Venue：日本大学理工学部 船橋キャンパス   Country：Japan  

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Venue：日本大学理工学部 船橋キャンパス   Country：Japan  

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Venue：日本大学理工学部 船橋キャンパス   Country：Japan  

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Venue：神戸ポートアイランド   Country：Japan  

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Venue：神戸ポートアイランド   Country：Japan  

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Language：Japanese   Presentation type：Poster presentation  

Venue：名古屋大学豊田講堂   Country：Japan  

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Venue：名古屋大学豊田講堂   Country：Japan  

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Venue：名古屋大学豊田講堂   Country：Japan  

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Venue：名古屋大学豊田講堂   Country：Japan  

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Venue：東京大学駒場キャンパス   Country：Japan  

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Venue：東京理科大学 野田キャンパス   Country：Japan  

Event date： 2023.9

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Venue：東京理科大学 野田キャンパス   Country：Japan  

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Venue：東京理科大学 野田キャンパス   Country：Japan  

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Venue：東京理科大学 野田キャンパス   Country：Japan  

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Venue：東京理科大学 野田キャンパス   Country：Japan  

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Venue：幕張メッセ国際会議場   Country：Japan  

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Venue：名古屋大学   Country：Japan  

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Venue：名古屋大学   Country：Japan  

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Venue：名古屋大学   Country：Japan  

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Venue：名古屋大学   Country：Japan  

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Venue：名古屋大学   Country：Japan  

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Event date： 2022.11 - 2022.12

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Event date： 2022.11 - 2022.12

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Event date： 2022.11

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Event date： 2022.9

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Event date： 2022.9

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Event date： 2022.7 - 2022.8

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Event date： 2022.3

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン  

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Venue：オンライン  

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Venue：オンライン  

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Venue：オンライン  

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Venue：オンライン  

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Venue：オンライン  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.10

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Venue：オンライン   Country：Japan  

Event date： 2021.9

Language：Japanese   Presentation type：Poster presentation  

Venue：オンライン   Country：Japan  

Event date： 2021.9

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Venue：オンライン   Country：Japan  

Event date： 2021.9

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Venue：オンライン   Country：Japan  

Event date： 2021.7

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Country：Japan  

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Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Poster presentation  

Venue：オンライン   Country：Japan  

Event date： 2021.6

Language：Japanese   Presentation type：Poster presentation  

Venue：オンライン   Country：Japan