Papers - MIYOSHI Daisuke
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Identification of Ligand-Responsive RNA G-Quadruplexes in the 3′ UTRs of Dengue Virus Serotypes Reviewed
Mohammad Jafar Sheikhi, Ayuka Onuma, Yutaro Imachi, Akira Shiraishi, Shoko Mori, Kohtaro Sugahara, Daisuke Miyoshi, Yue Ma, Takayuki Hishiki, Kazuo Nagasawa and Masayuki Tera
Biomolecules 2026.6
DOI： 10.3390/biom16070946
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Exclusion of RNA-binding domains from G-quadruplex condensates by G-quadruplex ligands Invited Reviewed International coauthorship
Yoshiki Hashimoto, Ryosuke Suzuki, Mizuho Aya, Nagisa Takamiya, Mitsuki Tsuruta,Takeru Torii, Toshiyuki Goto, Keiko Kawauchi and Daisuke Miyoshi
RSC Chemical Biology 2026.6
Authorship：Last author, Corresponding author
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Loss of p53 Provokes NF-κB-Dependent Disruption of Nucleolar Cap and Nucleoplasmic Redistribution of Fibrillarin During Nucleolar Stress
Takeru Torii, Mako Sumida, Atsushi Kobayashi, Toshiyuki Goto, Ryosuke Suzuki, Shin Kuwamoto, Wataru Nakajima, Wataru Sugimoto, Kohei Takeuchi, Yuma Tanaya, Masayuki Tera, Nobuyuki Tanaka, Hiroaki Hirata, Hisae Tateishi-Karimata, Takahito Nishikata, Miwako Kato Homma, Daisuke Miyoshi, Keiko Kawauchi
BIOMOLECULES 16 ( 2 ) 2026.2
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Investigation of the physicochemical and functional properties of poly(2-methacryloyloxyethyl phosphorylcholine)-conjugated aptamers. International journal
Seojung Cho, Jumpei Morimoto, Yutaro Saito, Yukiko Nagai, Asuka Sakata, Keitaro Yoshimoto, Mitsuki Tsuruta, Daisuke Miyoshi, Shinsuke Sando
Biomaterials science 14 ( 1 ) 232 - 239 2026.1
Polymer conjugation is a common strategy to improve the pharmacokinetics of aptamers, yet its effects on aptamer properties are incompletely understood. Poly(ethylene glycol) (PEG) is the most widely used polymer for this purpose, but concerns about anti-PEG immune responses have prompted interest in alternative polymers. We previously reported that conjugation with the zwitterionic polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) significantly prolongs the circulation time of a DNA aptamer while avoiding anti-PEG antibody recognition. In this study, we evaluated the physicochemical and functional consequences of PMPC conjugation of aptamers. Biophysical analyses suggested that the secondary structure and target-binding affinity of the aptamer were preserved, while functional consequences upon PMPC conjugation varied with the targets. The activity of a membrane receptor-targeting aptamer partially decreased, likely due to spatial constraints around the cell membrane, while RB005, targeting soluble activated coagulation factor IX, retained its full activity. In addition, PMPC conjugation significantly prolonged the in vivo plasma retention of RB005. By elucidating the effects of PMPC on aptamer properties and introducing another example that further supports the general applicability of PMPC conjugation in enhancing aptamer pharmacokinetics, these findings support PMPC as a promising alternative to PEG.
DOI： 10.1039/d5bm01078k
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Phase Separation of RX Repeat Peptides with Nucleic Acids. International journal
Sumit Shil, Mitsuki Tsuruta, Ryosuke Suzuki, Yoshiki Hashimoto, Takeru Torii, Shinya Taniguchi, Tomohiro Umetani, Keiko Kawauchi, Daisuke Miyoshi
Chemistry, an Asian journal 20 ( 23 ) e00805 2025.12
Biomolecular liquid-liquid phase separation (LLPS) plays a crucial role in organizing membraneless cellular compartments, which regulate a wide variety of cellular processes. A key molecular mechanism underlying LLPS of nucleic acids involves G-quadruplex (G4) structures of DNA and RNA interacting with intrinsically disordered proteins, particularly arginine and glycine (RGG/RG) rich proteins. The role of arginine residues in LLPS has been studied extensively, whereas few studies have focused on the role of the another frequently occurring residues, glycine. Here, we systematically investigated the contribution of G residues by substituting them with alanine (A), proline (P), valine (V), and tyrosine (Y) residues, generating a series of RX repeat peptides. Turbidity and microscopy assays with DNA oligonucleotides forming G4, duplex, as well as random coil, showed that RP and RA-peptides enhanced LLPS with G4 DNA, by comparing RG-peptide. In contrast, RY promoted liquid-solid phase separation (LSPS) with the G4 DNA, although it underwent LLPS with the random coil and duplex DNAs. In addition, RV-peptide formed aggregates even in the absence of any DNA. These results demonstrate that side-chain size, hydrophobicity, and aromaticity are critical factors for the LLPS and LSPS capability and selectivity with DNA forming various secondary structures. This study provides mechanistic insights into protein-nucleic acid LLPS and LSPS and guides the rational design peptides to undergo LLPS but not LSPS with nucleic acids.
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Liquid-liquid phase separation induced by i-motif DNA under molecular crowding conditions Invited Reviewed
Ryosuke Suzuki, Mitsuki Tsuruta, Sumit Shil, Kosei Morohashi, Keiko Kawauchi, Daisuke Miyoshia
Polymer J. 57 931 - 940 2025.4
Authorship：Corresponding author
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Three- and four-stranded nucleic acid structures and their ligands Invited Reviewed International journal
Yoshiki Hashimoto, Sumit Shil, Mitsuki Tsuruta, Keiko Kawauchi, and Daisuke Miyoshi
RSC Chemical Biology 6 ( 4 ) 466 - 491 2025.3
Authorship：Corresponding author
Nucleic acids have the potential to form not only duplexes, but also various non-canonical secondary structures in living cells. Non-canonical structures play regulatory functions mainly in the central dogma. Therefore, nucleic acid targeting molecules are potential novel therapeutic drugs that can target 'undruggable' proteins in various diseases. One of the concerns of small molecules targeting nucleic acids is selectivity, because nucleic acids have only four different building blocks. Three- and four-stranded non-canonical structures, triplexes and quadruplexes, respectively, are promising targets of small molecules because their three-dimensional structures are significantly different from the canonical duplexes, which are the most abundant in cells. Here, we describe some basic properties of the triplexes and quadruplexes and small molecules targeting the triplexes and tetraplexes.
DOI： 10.1039/D4CB00287C
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ケミカルバイオロジーの冒険（１２） 核酸を標的とした低分子・中分子化合物 Invited
堂野主税、勝田陽介、三好大輔
現代化学 648 32 - 39 2025.2
Authorship：Last author
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Roles of Loop Region in Folding Kinetics and Transcription Inhibition of DNA G-Quadruplexes Reviewed
Minori Nakata, Naoki Kosaka, Keiko Kawauchi, Daisuke Miyoshi
Biochemistry 64 609 - 619 2025.1
Authorship：Corresponding author
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Heterogeneity of Molecular Crowding and Liquid-Liquid Phase Separation. International journal
Mitsuki Tsuruta, Sumit Shil, Keiko Kawauchi, Daisuke Miyoshi
Sub-cellular biochemistry 109 327 - 345 2025
The inside of a living cell is highly crowded with extremely diverse biomacromolecules, small metabolites and osmolytes. The molecular conditions in cells change dynamically and rapidly depending on the cell cycle and state, organelle, and compartment. Much remains unknown regarding how biomolecular interactions and reactions can proceed in a spatiotemporally specific manner in such crowded, heterogeneous, and dynamic molecular environments. Selective condensation/droplet formation of biomolecules via liquid-liquid phase separation may be critical for interactions and reactions inside cells. In this chapter, we briefly describe the heterogeneity of molecular environments inside cells and the biological roles of liquid-liquid phase separation that allows biomolecular interactions and reactions in such heterogenous molecular environments. Finally, we discuss the mutual relationship between molecular crowding and liquid-liquid phase separation.
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NRAS DNA G-quadruplex-targeting molecules for sequence-selective enzyme inhibition Invited Reviewed
Yoshiki Hashimoto, Hiroki Kubo, Keiko Kawauchi and Daisuke Miyoshi
Chem. Commun. 60 13179 - 13182 2024.10
Authorship：Corresponding author
DOI： 10.1039/D4CC03753G
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The role of cytosine methylation in regulating the topology and liquid-liquid phase separation of DNA G-quadruplexes Reviewed International journal
Mitsuki Tsuruta, Sumit Shil, Shinya Taniguchi, Keiko Kawauchi and Daisuke Miyoshi
Chem. Sci. 16 ( 10 ) 4213 - 4225 2024.10
Authorship：Corresponding author
Aberrant expansion of GGGGCC DNA repeats that form G-quadruplexes (G4) is the main cause of amyotrophic lateral sclerosis (ALS). Expanded GGGGCC repeats induce liquid-liquid phase separation (LLPS) through their interaction with cellular proteins. Furthermore, GGGGCC expansion induces cytosine methylation (mC). Previous studies have shown that even slight chemical modifications of RNAs and proteins can drastically affect their LLPS ability, yet the relationship between LLPS and epigenetic DNA modifications like mC remains unexplored. As a model system, we investigated the effects of mC on LLPS induced by GGGGCC repeat DNAs and show for the first time that mC suppresses LLPS by altering the topology of G4 from being parallel to antiparallel.
DOI： 10.1039/D4SC06959E
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Factors Affecting Liquid-Liquid Phase Separation of RGG Peptides with DNA G-Quadruplex Invited Reviewed
Sumit Shil, Mitsuki Tsuruta, Keiko Kawauchi, Daisuke Miyoshi
ChemMedChem 2024.9
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Bioinformatic Analysis of Actin-Binding Proteins in the Nucleolus During Heat Shock Invited Reviewed International journal
Shinya Taniguchi, Takeru Torii ,Toshiyuki Goto, Kohei Takeuchi, Rine Katsumi, Mako Sumida, Sunmin Lee, Wataru Sugimoto, Masaya Gessho, Katsuhiko Itoh, Hiroaki Hirata, Junji Kawakami, Daisuke Miyoshi and Keiko Kawauchi
Genes 15 ( 12 ) 1580 2024.9
BACKGROUND/OBJECTIVES: Actin plays a crucial role not only in the cytoplasm, but also in the nucleus, influencing various cellular behaviors, including cell migration and gene expression. Recent studies reveal that nuclear actin dynamics is altered by cellular stresses, such as DNA damage; however, the effect of heat shock on nuclear actin dynamics, particularly in the nucleolus, remains unclear. This study aims to elucidate the contribution of nucleolar actin to cellular responses under heat shock conditions. METHODS: Nuclear actin dynamics in response to heat shock were investigated using nAC-GFP, a GFP-tagged actin chromobody, to visualize nuclear actin in HeLa cells. Bioinformatic analyses were also performed. RESULTS: Heat shock induced the reversible assembly of nAC-GFP in the nucleolus, with disassembly occurring upon recovery in a heat shock protein (Hsp) 70-dependent manner. Because the nucleolus, formed via liquid-liquid phase separation (LLPS), sequesters misfolded proteins under heat shock to prevent irreversible aggregation, we hypothesized that nucleolar actin-binding proteins might also be sequestered in a similar manner. Using several databases, we identified 47 actin-binding proteins localized in the nucleolus and determined the proportion of intrinsically disordered regions (IDRs) known to promote LLPS. Our analysis revealed that many of these 47 proteins exhibited high levels of IDRs. CONCLUSIONS: The findings from our bioinformatics analysis and further cellular studies may help elucidate new roles for actin in the heat shock response.
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Quantitative Effects of the Loop Region on Topology, Thermodynamics, and Cation Binding of DNA G-quadruplexes Reviewed International journal
Minori Nakata, Naoki Kosaka, Keiko Kawauchi, Daisuke Miyoshi
ACS Omega 9 ( 32 ) 35028 - 35036 2024.7
Authorship：Last author, Corresponding author
The thermal stability of G-quadruplexes is important for their biological roles. G-quadruplexes are stable in the presence of cations such as K+ and Na+ because these cations coordinate in the G-quartet of four guanine bases. It is well known that the number of G-quartets and the configuration of the guanine bases affect the binding affinity of the cation. Recently, structures formed in the loop regions connecting the guanine stretches have attracted significant attention, because the loop region affects G-quadruplex properties, such as topology, thermal stability, and interactions with proteins and small molecules. Considering these effects, the loop region can also affect the binding affinity of the cations. Here, we designed a series of G-quadruplex-forming DNA sequences that contain a hairpin in a loop region and investigated the effects of the sequence and structure of the loop region on the cation binding affinity as well as the thermal stability of the G-quadruplex as a whole. First, structural analysis of the DNA sequences showed that the hairpin at the loop plays a key role in determining G4 topology (strand orientation). Second, in the case of the G-quadruplexes with the hairpin-forming loop region, it was found that a longer loop length led to a higher thermodynamic stability of the G-quadruplex as well as higher cation binding affinity. In contrast, an unstructured loop region did not lead to such effects. Interestingly, the cation binding affinity was correlated to the thermodynamic stability of the hairpin structure at the loop region. It was quantitatively demonstrated that the stable loop region stabilized the whole G-quadruplex structure, which induced higher cation binding affinity. These systematic and quantitative results showed that the loop region is one of the determinants of cation binding and expanded the possibilities of drug development targeting G4s by stabilizing the loop region.
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細胞内環境で安定化する核酸構造を標的にした低分子薬の開発 Invited
橋本 佳樹, 川内 敬子, 三好 大輔
MEDCHEM NEWS (日本薬学会） 34 ( 1 ) 36 - 42 2024.4
Authorship：Corresponding author
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RNA 高次構造を標的とした光線力学療法の展望 Invited
取井猛流・木下菜月・橋本佳樹・杉本渉・川内敬子・三 好大輔
38 ( 13 ) 1154 - 1156 2023.11
Authorship：Last author, Corresponding author
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Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments Reviewed International journal
Takeru Torii, Wataru Sugimoto, Katsuhiko Itoh, Natsuki Kinoshita, Masaya Gessho, Toshiyuki Goto, Ikuno Uehara, Wataru Nakajima, Yemima Budirahardja, Daisuke Miyoshi, Takahito Nishikata, Nobuyuki Tanaka, Hiroaki Hirata, Keiko Kawauchi
Cell Death & Disease 14 ( 11 ) 766766 - 766 2023.8
Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.
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The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells Invited Reviewed International journal
Natsuki Kinoshita, Masaya Gessho, Takeru Torii, Yukako Ashida, Minori Akamatsu, Alvin Kunyao Guo, Sunmin Lee, Tatsuya Katsuno 3, Wataru Nakajima Yemima Budirahardja, Daisuke Miyoshi, Takehiko Todokoro, Hiroki Ishida, Takahito Nishikata, Keiko Kawauchi
Genes Cells 28 ( 9 ) 653 - 662 2023.6
Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.
DOI： 10.1111/gtc.13053
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Quantification of the concentration in a droplet formed by liquid–liquid phase separation of G-quadruplex-forming RNA Reviewed
Kohei Yokosawa, Mitsuki Tsuruta, Shinji Kajimoto, Naoki Sugimoto, Daisuke Miyoshi, Takakazu Nakabayashi
Chem. Phys. Lett. 826 140634 - 140634 2023.6