論文 - 杉本 直己
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Imperfect G-quadruplex as an emerging candidate for transcriptional regulation 査読あり
S. Sarkar, H. Tateishi-Karimata, T. Ohyama, and N. Sugimoto
Nucleic Acids Res. 53 ( 5 ) gkaf164 - gkaf164 2025年3月
DOI： 10.1093/nar/gkaf164
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Controlling the local conformation of RNA G-quadruplex results in reduced RNA/peptide cytotoxic accumulation associated with C9orf72 ALS/FTD 査読あり
S. Matsumoto, H. Tateishi-Karimata, T. Ohyama, and N. Sugimoto
Small Methods 9 2401630 - 2401630 2025年
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S. Ghosh, S. Takahashi, T. Ohyama, L. Liu, and N. Sugimoto
Journal of the American Chemical Society 146 ( 47 ) 32479 - 32497 2024年11月
担当区分：筆頭著者
DOI： 10.1021/jacs.4c09388
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Contrasting effect of different crowding agents on pseudoknot RNA stability 査読あり
S. Satpathi, T. Endoh and N. Sugimoto
Medicinal Chemistry Research 33 2079 - 2084 2024年8月
Pseudoknot (PK) is one of the prevalent and potential targets for drug discovery. It is still difficult to predict PK-forming regions within endogenous RNA transcripts because the intracellular environment consisting of various cellular agents has diverse effects on the structure and stability of nucleic acids. In this study, we have demonstrated the impact of the varied crowding conditions on the structural stability of pseudoknot RNA, emphasizing the distinct stabilities of PK structures depending on the crowding environment.
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Guanidine modification improves functions of natural RNA-targeting alkaloids 査読あり 国際共著
Tamaki Endoh, Sagar Satpathi, Yutong Chen, Saki Matsumoto, Tatsuya Ohyama, Peter Podbevšek, Janez Plavec, Kazumitsu Onizuka, Fumi Nagatsugi, Naoki Sugimoto
New Journal of Chemistry 48 ( 19 ) 8529 - 8533 2024年4月
出版者・発行元：Royal Society of Chemistry (RSC)
Guanidine modification on berberine improved RNA binding affinity and enhanced suppression of protein expression.
DOI： 10.1039/d3nj05833f
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Hisae Tateishi-Karimata, Keiko Kawauchi, Shuntaro Takahashi, and Naoki Sugimoto
J. Am. Chem. Soc., 146 ( 12 ) 8005 - 8015 2024年3月
共著
出版者・発行元：American Chemical Society (ACS)
Intracellular chemical microenvironments, including ion concentrations and molecular crowding, play pivotal roles in cell behaviors, such as proliferation, differentiation, and cell death via regulation of gene expression. However, there is no method for quantitative analysis of intracellular environments due to their complexity. Here, we have developed a system for highlighting the environment inside of the cell (SHELL). SHELL is a pseudocellular system, wherein small molecules are removed from the cell and a crowded intracellular environment is maintained. SHELL offers two prominent advantages: (1) It allows for precise quantitative biochemical analysis of a specific factor, and (2) it enables the study of any cell, thereby facilitating the study of target molecule effects in various cellular environments. Here, we used SHELL to study G-quadruplex formation, an event that implicated cancer. We show that G-quadruplexes are more stable in SHELL compared with in vitro conditions. Although malignant transformation perturbs cellular K+ concentrations, environments in SHELL act as buffers against G-quadruplex destabilization at lower K+ concentrations. Notably, the buffering effect was most pronounced in SHELL derived from nonaggressive cancer cells. Stable G-quadruplexes form due to the binding of the G-quadruplex with K+ in different cancer cells. Furthermore, the observed pattern of G-quadruplex-induced transcriptional inhibition in SHELL is consistent with that in living cells at different cancer stages. Our results indicate that ion binding to G-quadruplexes regulates gene expression during pathogenesis.
DOI： 10.1021/jacs.3c11160
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Hisae Tateishi-Karimata, Naoki Sugimoto
ACS Omega 9 ( 5 ) 5675 - 5682 2024年1月
G-quadruplexes in disease-related genes are associated with various biological processes and regulate disease progression. Although methods involving ligands and other techniques are available to stabilize G-quadruplexes, approaches for destabilizing G-quadruplexes remain limited. Here, we evaluated whether G-quadruplexes can be destabilized using choline dihydrogen phosphate (choline dhp), a highly biocompatible hydrated ionic liquid. Circular dichroism spectral measurements at increasing temperatures revealed that choline dhp destabilized G-quadruplexes more effectively than did KCl-containing solutions. Thermodynamic analysis indicated that destabilization occurred via an entropic contribution, suggesting that choline ions did not coordinate with the G-quartets, because of their large radii. Subsequently, plasmid DNAs containing G-quadruplexes were constructed, and transcription reactions were performed in nuclear extracts from living cells. G-quadruplexes repressed transcription, whereas the addition of choline dhp increased transcription. Although ionic liquids often inactivate biomolecules, choline dhp can be used to culture various cells. Furthermore, the transcription of template DNA containing the G-quadruplex was greatly enhanced in living MDA-MD-231 cells (aggressive human breast cancer cells) cultured with choline dhp. Our results show that choline dhp destabilizes G-quadruplexes in cells, indicating that choline dhp can regulate gene expression. Thus, choline dhp may be useful for regulating target disease-related genes.
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In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics. 査読あり 国際共著 国際誌
Dipanwita Banerjee, Hisae Tateishi-Karimata, Maria Toplishek, Tatsuya Ohyama, Saptarshi Ghosh, Shuntaro Takahashi, Marko Trajkovski, Janez Plavec, Naoki Sugimoto
Journal of the American Chemical Society 145 ( 43 ) 23503 - 23518 2023年11月
In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.
DOI： 10.1021/jacs.3c06706
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Theranostic approach to specifically targeting the interloop region of BCL2 i-motif DNA by crystal violet 査読あり 国際共著 国際誌
Sinjan Das , Shuntaro Takahashi , Tatsuya Ohyama, Sudipta Bhowmik , Naoki Sugimoto
Scientific Reports 13 2023年9月
Ligands that recognise specific i-motif DNAs are helpful in cancer diagnostics and therapeutics, as i-motif formation can cause cancer. Although the loop regions of i-motifs are promising targets for ligands, the interaction between a ligand and the loop regions based on sequence information remains unexplored. Herein, we investigated the loop regions of various i-motif DNAs to determine whether these regions specifically interact with fluorescent ligands. Crystal violet (CV), a triphenylmethane dye, exhibited strong fluorescence with the i-motif derived from the promoter region of the human BCL2 gene in a sequence- and structure-specific manner. Our systematic sequence analysis indicated that CV was bound to the site formed by the first and third loops through inter-loop interactions between the guanine bases present in these loops. As the structural stability of the BCL2 i-motif was unaffected by CV, the local stabilisation of the loops by CV could inhibit the interaction of transcription factors with these loops, repressing the BCL2 expression of MCF-7 cells. Our finding suggests that the loops of the i-motif can act as a novel platform for the specific binding of small molecules; thus, they could be utilised for the theranostics of diseases associated with i-motif DNAs.
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Kohei Yokosawa, Mitsuki Tsuruta, Shinji Kajimoto, Naoki Sugimoto, Daisuke Miyoshi, Takakazu Nakabayashi
Chemical Physics Letters 826 140634 2023年9月
出版者・発行元：Elsevier BV
Liquid–liquid phase separation (LLPS), which results in the formation of highly concentrated droplets of biomolecules, is involved in various physiological phenomena. We have performed a label-free quantification of the concentration of a G-quadruplex-forming RNA and an RGG peptide in a single droplet using Raman microscopy. The concentration ratio of the RNA to the peptide within the droplet was found to maintain even when the prepared concentration ratio of these two species was varied. This result indicates that electrostatic interactions between the RNA and the peptide induced the droplet formation. It was also shown that the RNA maintains its G-quadruplex structure inside the droplets.
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S. Ghosh, S. Takahashi, D. Banerjee, T. Ohyama, T. Endoh, H. Tateishi-Karimata, and N. Sugimoto
Nucleic Acids Res. 51 4101 - 4111 2023年5月
RNA performs various spatiotemporal functions in living cells. As the solution environments significantly affect the stability of RNA duplexes, a stability prediction of the RNA duplexes in diverse crowded conditions is required to understand and modulate gene expression in heterogeneously crowded intracellular conditions. Herein, we determined the nearest-neighbor (NN) parameters for RNA duplex formation when subjected to crowding conditions with an ionic concentration relevant to that found in cells. Determination of the individual contributions of excluded volume effect and water activity to each of the NN parameters in crowded environments enabled prediction of the thermodynamic parameters and their melting temperatures for plenty of tested RNA duplex formation in vitro and in cell with significant accuracy. The parameters reported herein will help predicting RNA duplex stability in different crowded environments, which will lead to an improved understanding of the stability-function relationship for RNAs in various cellular organelles with different molecular environments.
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Simple and fast screening for structure-selective G-quadruplex ligands 査読あり 国際誌
Y. Hashimoto, Y. Imagawa, K. Nagano, R. Maeda, N. N.agahama, T. Torii, N. Kinoshita, N. Takamiya, K. Kawauchi, H. Tateishi-Karimata, N. Sugimoto and D. Miyoshi
Chem. Commun. 59 4891 - 4894 2023年4月
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Endogenous G-quadruplex-forming RNAs inhibit the activity of SARS-CoV-2 RNA polymerase 査読あり 国際誌
T. Endoh, S. Takahashi, and N. Sugimoto
Chem. Commun. 59 872 - 875 2023年1月
Replication of RNA viruses is catalysed by virus-specific polymerases, which can be targets of therapeutic strategies. In this study, we used a selection strategy to identify endogenous RNAs from a transcriptome library derived from lung cells that interact with the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2. Some of the selected RNAs weakened the activity of RdRp by forming G-quadruplexes. These results suggest that certain endogenous RNAs, which potentially form G-quadruplexes, can reduce the replication of viral RNAs.
DOI： 10.1039/d2cc05858h
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Pressure-temperature control of activity of RNA polymerase ribozyme 査読あり 国際誌
S. Takahashi and N. Sugimoto
Biophys. Chem. 292 106914 2023年1月
A representative role of nucleic acids (DNA and RNA) is in the storage of genetic information. In contrast, RNAs act as ribozymes that catalyze various biochemical reactions. The “RNA world” hypothesis suggests that the origin of life was RNA because a ribozyme that shows RNA replication activity has been identified. However, prebiotic conditions in the RNA world remain unknown. In this study, we investigated the effect of high pressure and temperature on RNA replication using an RNA polymerase ribozyme tC9Y. We found that pressure accelerated the RNA replication activity of tC9Y ribozyme at higher temperatures than physiological conditions. Furthermore, molecular crowding by concentrated polyethylene glycol 200 (average molecular weight 200) synergistically enhanced the replication activity at higher pressure and temperature because the negative effect of a volumetric contribution of hydration on the tC9Y ribozyme activity decreased under crowding conditions. As a comparison, proteinaceous RNA polymerase that exists in the modern era did not show accelerated activity under high pressure and temperature. Thus, these results imply that the prebiotic conditions for the RNA world were at high pressure and temperatures under crowding conditions.
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Cladogenetic Orthogonal Light-Up Aptamers for Simultaneous Detection of Multiple Small Molecules in Cells 査読あり 国際共著 国際誌
T. Endoh, J. H. Tan, S. B. Chen, and N. Sugimoto
Anal. Chem. 95 976 - 985 2022年12月
Recent successes in construction of light-up RNA aptamers allowed fluorescence-based live-cell imaging of RNAs. In addition, light-up aptamers have been converted into signaling aptamers that enable fluorometric detection of small chemicals. To date, only a single target chemical has been detected at a time in cells. In this study, we selected cladogenetic orthogonal light-up aptamers that output three different colors from the RNA library having the same ligand binding core. Two of the three functioned in mammalian cells. These two aptamers, which fluoresce blue and green upon binding of cognate fluorogen, were converted into signaling aptamers. Using these signaling aptamers in combination with a previously described light-up aptamer with red fluorescence, we demonstrated simultaneous detection of multiple chemicals in living cells. The cladogenetic orthogonal light-up aptamers developed in this study and the simple strategy for rational designing of the signaling aptamers will provide innovative advances in the field of RNA-based bioimaging.
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High-temperature adaptation of an OsNRT2.3 allele is thermoregulated by small RNAs 査読あり 国際共著 国際誌
Y. Zhang, H. Tateishi-Karimata, T. Endoh, Q. J., Kexin Li, X. Fan, Y. Ma, L. Gao, H. Lu, Z. Wang, A. E. Cho, X. Yao, Ch. Liu, N. Sugimoto, S. Guo, X. Fu, Q. Shen, G. Xu, L. R. Herrera-Estrella, X. Fan
Science Advances 8 9785 2022年11月
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Controlling liquid-liquid phase separation of G-quadruplex-forming RNAs in a sequence-specific manner. 査読あり 国際誌
Mitsuki Tsuruta, Takeru Torii, Kazuki Kohata, Keiko Kawauchi, Hisae Tateishi-Karimata, Naoki Sugimoto, Daisuke Miyoshi
Chemical communications (Cambridge, England) 58 ( 93 ) 12931 - 12934 2022年11月
We constructed a minimum liquid-liquid phase separation model system to form liquid droplets using only G-quadruplex-forming oligonucleotides and R- and G-rich oligopeptides. We found that the G-quadruplex structure is an essential component for RNA to form droplets with the peptide. Based on this model system and our findings, droplet redissolution via structure transition from a G-quadruplex to a duplex was achieved in a sequence-specific manner.
Liquid–liquid phase separation (LLPS) of biomacromolecules has been attracting attention as a novel phenomenon and a new phase in living cells.1 It is now evident that LLPS involving nucleic acids participates in the regulation of gene expression at various levels, including replication, transcription, RNA processing, localization, and translation.2 Dysfunction of LLPS is linked to the onset of viral infection,3 cancer,4 and neurodegenerative diseases.2 Although LLPS is induced by different molecular compositions, it is considered that LLPS of different molecular compositions has common assembly and disassembly mechanisms.5 Many of the protein motifs essential for LLPS are intrinsically disordered regions (IDRs) such as RGG motifs.6 Moreover, databases and prediction models of proteins undergoing LLPS have been established.7 Furthermore, fundamental properties of RNAs for inducing LLPS is becoming clear.8–11 RNA secondary structure-dependent LLPS has also been reported.12
Of note, the RGG domain derived from various RNA binding proteins (RBPs) selectively binds with G-quadruplexes (G4s), a non-canonical secondary structure of nucleic acids.13 G4 formed by guanine-rich sequences have been reported to induce LLPS in a structure-specific manner. Fragile X mental retardation protein (FMRP),14 SERPINE1 mRNA-binding protein 1 (SERBP1),15 and Histone H116 all induce LLPS with G4. The repetitive GGGGCC sequence, found within the C9orf72 gene, undergoes pathogenic expansion responsible for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).17 Repetitive expansion of GGC repeats on the FMR1 gene is considered as an onset mechanism of neurodegenerative diseases such as fragile X syndrome (FXS) and fragile X-associated tremor/ataxia syndrome (FXTAS).18 These sequences can fold to form G4 structures and induce aberrant RNA foci in the nucleus through sequestration of various RBPs.19 Although the characteristics of the various protein and nucleic acid sequences of droplets are becoming clearer, methods for regulating droplets have not yet been developed. Here, we developed a model system of LLPS based on oligonucleotides and oligopeptides to investigate the essential properties of RNA that directly induce and control LLPS.
A series of RNA oligonucleotides were designed to study their ability to undergo LLPS with a cationic peptide containing the RGG motif derived from FMRP (Table 1).13 FMR1 RNA and C9orf72 RNA are four repeats of GGC and GGGGCC, respectively, which may form a G4. sFMR1 RNA is a scrambled sequence of FMR1 RNA that does not form a G4. Comp-RNA is complementary to FMR1 RNA. dsRNA was designed to form an intramolecular duplex (a hairpin loop structure). The structure of the RNA oligonucleotides was studied by CD spectroscopy and UV melting curves (Fig. S1, ESI†). These results are consistent with each other and show that FMR1 RNA and c9orf72 RNA fold into parallel G4s, and the 1 : 1 mixture and dsRNA forms A-form duplexes in the experimental conditions. (See Fig. S1, ESI† for explanations of the CD spectra and UV melting curves).DOI： 10.1039/d2cc04366a
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DNA methylation is regulated by both the stability and topology of G-quadruplex 査読あり 国際誌
S. Matsumoto, H. Tateishi-Karimata, N. Sugimoto
Chem. Commun., 58 12459 - 12462 2022年10月
DOI： 10.1039/d2cc04383a
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Volumetric strategy for quantitatively elucidating local hydration network around a G-quadruplex 査読あり 国際共著 国際誌
S. Matsumoto, S. Takahashi, S. Bhowmik, T. Ohyama, and N. Sugimoto
Anal. Chem 94 ( Selected as a Supplemental Cover ) 7400 - 7407 2022年5月
担当区分：最終著者
Hydration around nucleic acids, such as DNA and RNA, is an important factor not only for the stability of nucleic acids but also for their interaction with binding molecules. Thus, it is necessary to quantitatively elucidate the hydration properties of nucleic acids around a certain structure. In this study, volumetric changes in G-quadruplex (G4) RNA formation were investigated by systematically changing the number of G-quartet stacks under high pressure. The volumetric contribution at the level of each G4 structural unit revealed that the core G4 helix was significantly more dehydrated than the other parts, including the edges of G-quartets and loops. These findings will help in predicting the binding of G4 ligands on the surface of G4, depending on the chemical structure of the ligand and solution environment. Therefore, the preset volumetric parameter provides information that can predict molecular interactions in G4 formations during molecular crowding in cells.
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Applicability of Nearest-neighbour Model for Pseudoknot RNAs 査読あり 国際誌
S. Satpathi, T. Endoh, and N. Sugimoto
Chem. Commun 58 ( [Selected as an Inside Back Cover] ) 5952 - 5955 2022年4月
担当区分：最終著者
DOI： 10.1039/d1cc07094k