Papers - TAKAHASHI Shuntaro
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Elucidating the role of groove hydration on stability and functions of biased DNA duplexes in cell-like chemical environments Reviewed
Saptarshi Ghosh, Shuntaro Takahashi, Tatsuya Ohyama, Lutan Liu, Naoki Sugimoto
Journal of the american chemical society 2024.11
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Development of a pseudocellular system to quantify specific interactions determining the g-quadruplex function in cells Reviewed
Hisae Tateishi-Karimata, Keiko Kawauchi, Shuntaro Takahashi, Naoki Sugimoto
Journal of the american chemical society 2024.3
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In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics. Reviewed International coauthorship International journal
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 2023.11
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. Reviewed International coauthorship International journal
Sinjan Das, Shuntaro Takahashi, Tatsuya Ohyama, Sudipta Bhowmik, Naoki Sugimoto
Scientific reports 13 ( 1 ) 14338 - 14338 2023.9
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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Nearest-neighbor parameters for the prediction of RNA duplex stability in diverse in vitro and cellular-like crowding conditions. Reviewed International journal
Saptarshi Ghosh, Shuntaro Takahashi, Dipanwita Banerjee, Tatsuya Ohyama, Tamaki Endoh, Hisae Tateishi-Karimata, Naoki Sugimoto
Nucleic acids research 51 ( 9 ) 4101 - 4111 2023.5
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.
DOI： 10.1093/nar/gkad020
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Endogenous G-quadruplex-forming RNAs inhibit the activity of SARS-CoV-2 RNA polymerase. Reviewed International journal
Tamaki Endoh, Shuntaro Takahashi, Naoki Sugimoto
Chemical communications (Cambridge, England) 59 ( 7 ) 872 - 875 2023.1
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. Reviewed International journal
Shuntaro Takahashi, Naoki Sugimoto
Biophysical chemistry 292 106914 - 106914 2022.10
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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Volumetric Strategy for Quantitatively Elucidating a Local Hydration Network around a G-Quadruplex. Reviewed International coauthorship International journal
Saki Matsumoto, Shuntaro Takahashi, Sudipta Bhowmik, Tatsuya Ohyama, Naoki Sugimoto
Analytical chemistry 94 ( 20 ) 7400 - 7407 2022.5
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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Replication Control of Human Telomere G-Quadruplex DNA by G-Quadruplex Ligands Dependent on Solution Environment Reviewed International coauthorship
Shuntaro Takahashi, Sudipta Bhowmik, Shinobu Sato, Shigeori Takenaka, Naoki Sugimoto
Life 12 ( 4 ) 553 - 553 2022.4
Publisher：{MDPI} {AG}
The human telomere region is known to contain guanine-rich repeats and form a guanine-quadruplex (G4) structure. As telomeres play a role in the regulation of cancer progression, ligands that specifically bind and stabilize G4 have potential therapeutic applications. However, as the human telomere sequence can form G4 with various topologies due to direct interaction by ligands and indirect interaction by the solution environment, it is of great interest to study the topology-dependent control of replication by ligands. In the present study, a DNA replication assay of a template with a human telomere G4 sequence in the presence of various ligands was performed. Cyclic naphthalene diimides (cNDI1 and cNDI2) efficiently increased the replication stall of the template DNA at G4 with an anti-parallel topology. This inhibition was stability-dependent and topology-selective, as the replication of templates with hybrid or parallel G4 structures was not affected by the cNDI and cNDI2. Moreover, the G4 ligand fisetin repressed replication with selectivity for anti-parallel and hybrid G4 structures without stabilization. Finally, the method used, referred to as quantitative study of topology-dependent replication (QSTR), was adopted to evaluate the correlation between the replication kinetics and the stability of G4. Compared to previous results obtained using a modified human telomere sequence, the relationship between the stability of G4 and the effect on the topology-dependent replication varied. Our results suggest that native human telomere G4 is more flexible than the modified sequence for interacting with ligands. These findings indicate that the modification of the human telomeric sequence forces G4 to rigidly form a specific structure of G4, which can restrict the change in topology-dependent replication by some ligands.
DOI： 10.3390/life12040553
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Ruthenium Polypyridyl Complex Bound to a Unimolecular Chair-Form G-Quadruplex Reviewed International coauthorship International journal
Kane T. McQuaid, Shuntaro Takahashi, Lena Baumgaertner, David J. Cardin, Neil G. Paterson, James P. Hall, Naoki Sugimoto, Christine J. Cardin
Journal of the American Chemical Society 144 ( 13 ) 5956 - 5964 2022.4
Publisher：American Chemical Society ({ACS})
The DNA G-quadruplex is known for forming a range of topologies and for the observed lability of the assembly, consistent with its transient formation in live cells. The stabilization of a particular topology by a small molecule is of great importance for therapeutic applications. Here, we show that the ruthenium complex Λ-[Ru(phen)2(qdppz)]2+ displays enantiospecific G-quadruplex binding. It crystallized in 1:1 stoichiometry with a modified human telomeric G-quadruplex sequence, GGGTTAGGGTTAGGGTTTGGG (htel21T18), in an antiparallel chair topology, the first structurally characterized example of ligand binding to this topology. The lambda complex is bound in an intercalation cavity created by a terminal G-quartet and the central narrow lateral loop formed by T10-T11-A12. The two remaining wide lateral loops are linked through a third K+ ion at the other end of the G-quartet stack, which also coordinates three thymine residues. In a comparative ligand-binding study, we showed, using a Klenow fragment assay, that this complex is the strongest observed inhibitor of replication, both using the native human telomeric sequence and the modified sequence used in this work.
DOI： 10.1021/jacs.2c00178
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Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase. Reviewed International journal
Shuntaro Takahashi, Saki Matsumoto, Pallavi Chilka, Saptarshi Ghosh, Hiromichi Okura, Naoki Sugimoto
Scientific reports 12 ( 1 ) 1149 - 1149 2022.1
In biological systems, the synthesis of nucleic acids, such as DNA and RNA, is catalyzed by enzymes in various aqueous solutions. However, substrate specificity is derived from the chemical properties of the residues, which implies that perturbations of the solution environment may cause changes in the fidelity of the reaction. Here, we investigated non-promoter-based synthesis of RNA using T7 RNA polymerase (T7 RNAP) directed by an RNA template in the presence of polyethylene glycol (PEG) of various molecular weights, which can affect polymerization fidelity by altering the solution properties. We found that the mismatch extensions of RNA propagated downstream polymerization. Furthermore, PEG promoted the polymerization of non-complementary ribonucleoside triphosphates, mainly due to the decrease in the dielectric constant of the solution. These results indicate that the mismatch extension of RNA-dependent RNA polymerization by T7 RNAP is driven by the stacking interaction of bases of the primer end and the incorporated nucleotide triphosphates (NTP) rather than base pairing between them. Thus, proteinaceous RNA polymerase may display different substrate specificity with changes in dielectricity caused by molecular crowding conditions, which can result in increased genetic diversity without proteinaceous modification.
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Correction to 'Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition'. Reviewed International journal
Dipanwita Banerjee, Hisae Tateishi-Karimata, Tatsuya Ohyama, Saptarshi Ghosh, Tamaki Endoh, Shuntaro Takahashi, Naoki Sugimoto
Nucleic acids research 49 ( 18 ) 10796 - 10799 2021.10
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Chemical Modulation of DNA Replication along G-Quadruplex Based on Topology-Dependent Ligand Binding. Reviewed International journal
Shuntaro Takahashi, Anita Kotar, Hisae Tateishi-Karimata, Sudipta Bhowmik, Zi-Fu Wang, Ta-Chau Chang, Shinobu Sato, Shigeori Takenaka, Janez Plavec, Naoki Sugimoto
Journal of the American Chemical Society 143 ( 40 ) 16458 - 16469 2021.9
Ligands that bind to and stabilize guanine-quadruplex (G4) structures to regulate DNA replication have therapeutic potential for cancer and neurodegenerative diseases. Because there are several G4 topologies, ligands that bind to their specific types may have the ability to preferentially regulate the replication of only certain genes. Here, we demonstrated that binding ligands stalled the replication of template DNA at G4, depending on different topologies. For example, naphthalene diimide derivatives bound to the G-quartet of G4 with an additional interaction between the ligand and the loop region of a hybrid G4 type from human telomeres, which efficiently repressed the replication of the G4. Thus, these inhibitory effects were not only stability-dependent but also topology-selective based on the manner in which G4 structures interacted with G4 ligands. Our original method, referred to as a quantitative study of topology-dependent replication (QSTR), was developed to evaluate correlations between replication rate and G4 stability. QSTR enabled the systematic categorization of ligands based on topology-dependent binding. It also demonstrated accuracy in determining quantitatively how G4 ligands control the intermediate state of replication and the kinetics of G4 unwinding. Hence, the QSTR index would facilitate the design of new drugs capable of controlling the topology-dependent regulation of gene expression.
DOI： 10.1021/jacs.1c05468
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Naoki Sugimoto, Tamaki Endoh, Shuntaro Takahashi, Hisae Tateishi-Karimata
Bulletin of the Chemical Society of Japan 94 ( 7 ) 1970 - 1998 2021.7
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Watson-Crick versus Hoogsteen Base Pairs: Chemical Strategy to Encode and Express Genetic Information in Life. Invited Reviewed
Shuntaro Takahashi, Naoki Sugimoto
Accounts of chemical research 2021.2
Joint Work
ConspectusNucleic acids typically form a double helix structure through Watson-Crick base-pairing. In contrast, non-Watson-Crick base pairs can form other three-dimensional structures. Although it is well-known that Watson-Crick base pairs may be more unstable than non-Watson-Crick base pairs under some conditions, the importance of non-Watson-Crick base pairs has not been widely examined. Hoogsteen base pairs, the non-Watson-Crick base pairs, contain important hydrogen-bond patterns that form the helices of nucleic acids, such as in Watson-Crick base pairs, and can form non-double helix structures such as triplexes and quadruplexes. In recent years, non-double helix structures have been discovered in cells and were reported to considerably influence gene expression. The complex behavior of these nucleic acids in cells is gradually being revealed, but the underlying mechanisms remain almost unknown.Quantitatively analyzing the structural stability of nucleic acids is important for understanding their behavior. A nucleic acid is an anionic biopolymer composed of a sugar, base, and phosphoric acid. The physicochemical factors that determine the stability of nucleic acid structures include those derived from the interactions of nucleic acid structures and those derived from the environments surrounding nucleic acids. The Gibbs free energy change (ΔG) of structure formation is the most commonly used physicochemical parameter for analyzing quantitative stability. Quantitatively understanding the intracellular behavior of nucleic acids involves describing the formation of nucleic acid structures and related reactions as ΔG. Based on this concept, we quantitatively analyzed the stability of double helix and non-double helix structures and found that decreased water activity, an important factor in crowded cellular conditions, significantly destabilize the formation of Watson-Crick base pairs but stabilizes Hoogsteen base pairs.Here, we describe a physicochemical approach to understand the regulation of gene expressions based on the stability of nucleic acid structures. We developed new methods for predicting the stability of double and non-double helices in various molecular environments by mimicking intracellular environments. Furthermore, the physicochemical approach used for analyzing gene expression regulated by non-double helix structures is useful for not only determining how gene expression is controlled by cellular environments but also for developing new technologies to chemically regulate gene expression by targeting non-double helix structures. We discuss the roles of Watson-Crick and Hoogsteen base pairs in cells based on our results and why both types of base pairing are required for life. Finally, a new concept in nucleic acid science beyond that of Watson and Crick base pairing is introduced.
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Engineering exosome polymer hybrids by atom transfer radical polymerization. Reviewed International coauthorship
Sushil Lathwal, Saigopalakrishna S Yerneni, Susanne Boye, Upenyu L Muza, Shuntaro Takahashi, Naoki Sugimoto, Albena Lederer, Subha R Das, Phil G Campbell, Krzysztof Matyjaszewski
Proceedings of the National Academy of Sciences of the United States of America 118 ( 2 ) 2021.1
Joint Work
Exosomes are emerging as ideal drug delivery vehicles due to their biological origin and ability to transfer cargo between cells. However, rapid clearance of exogenous exosomes from the circulation as well as aggregation of exosomes and shedding of surface proteins during storage limit their clinical translation. Here, we demonstrate highly controlled and reversible functionalization of exosome surfaces with well-defined polymers that modulate the exosome's physiochemical and pharmacokinetic properties. Using cholesterol-modified DNA tethers and complementary DNA block copolymers, exosome surfaces were engineered with different biocompatible polymers. Additionally, polymers were directly grafted from the exosome surface using biocompatible photo-mediated atom transfer radical polymerization (ATRP). These exosome polymer hybrids (EPHs) exhibited enhanced stability under various storage conditions and in the presence of proteolytic enzymes. Tuning of the polymer length and surface loading allowed precise control over exosome surface interactions, cellular uptake, and preserved bioactivity. EPHs show fourfold higher blood circulation time without altering tissue distribution profiles. Our results highlight the potential of precise nanoengineering of exosomes toward developing advanced drug and therapeutic delivery systems using modern ATRP methods.
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Stability prediction of canonical and non-canonical structures of nucleic acids in various molecular environments and cells. Reviewed
Shuntaro Takahashi, Naoki Sugimoto
Chemical Society reviews 2020.10
Joint Work
Nucleic acids (DNA and RNA) dynamically fold and unfold to exert their functions in cells. These folding and unfolding behaviours are also the basis for various technical applications. To understand the biological mechanism of nucleic acid function, and design active materials using nucleic acids, biophysical approaches based on thermodynamics are very useful. Methods for predicting the stability of canonical duplexes of nucleic acids have been extensively investigated for more than half a century and are now widely used. However, such predictions are not always accurate under various solution conditions, particularly cellular conditions, as the concentrations of cations and cosolutes under intracellular conditions, named as molecular crowding, differ from those under standard experimental conditions. Moreover, the crowding condition in cells is spatiotemporally variable. Furthermore, non-canonical structures such as triplex and tetraplex exist in cells and play important roles in gene expression. Therefore, a prediction method reflecting the cellular conditions must be established to determine the stability of various nuclei acid structures. This article reviews the biophysicochemical background of predicting nucleic acid stability and recent advances in the prediction of this stability under cellular conditions.
DOI： 10.1039/d0cs00594k
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Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates. Invited Reviewed International coauthorship
Shuntaro Takahashi, Piet Herdwijn, Naoki Sugimoto
Molecules (Basel, Switzerland) 25 ( 18 ) 2020.9
Joint Work
Unnatural nucleic acids are promising materials to expand genetic information beyond the natural bases. During replication, substrate nucleotide incorporation should be strictly controlled for optimal base pairing with template strand bases. Base-pairing interactions occur via hydrogen bonding and base stacking, which could be perturbed by the chemical environment. Although unnatural nucleobases and sugar moieties have undergone extensive structural improvement for intended polymerization, the chemical environmental effect on the reaction is less understood. In this study, we investigated how molecular crowding could affect native DNA polymerization along various templates comprising unnatural nucleobases and sugars. Under non-crowding conditions, the preferred incorporation efficiency of pyrimidine deoxynucleotide triphosphates (dNTPs) by the Klenow fragment (KF) was generally high with low fidelity, whereas that of purine dNTPs was the opposite. However, under crowding conditions, the efficiency remained almost unchanged with varying preferences in each case. These results suggest that hydrogen bonding and base-stacking interactions could be perturbed by crowding conditions in the bulk solution and polymerase active center during transient base pairing before polymerization. This study highlights that unintended dNTP incorporation against unnatural nucleosides could be differentiated in cases of intracellular reactions.
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Molecular crowding induces primer extension by RNA polymerase through base stacking beyond Watson-Crick rules Reviewed
Shuntaro Takahashi, Hiromichi Okura, Pallavi Chilka, Saptarshi Ghosh, Naoki Sugimoto
RSC Advances 10 ( 55 ) 33052 - 33058 2020.9
Joint Work
© The Royal Society of Chemistry. The polymerisation of nucleic acids is essential for copying genetic information correctly to the next generations, whereas mispolymerisation could promote genetic diversity. It is possible that in the prebiotic era, polymerases might have used mispolymerisation to accelerate the diversification of genetic information. Even in the current era, polymerases of RNA viruses frequently cause mutations. In this study, primer extension under different molecular crowding conditions was measured using T7 RNA polymerase as a model for the reaction in the prebiotic world. Interestingly, molecular crowding using 20 wt% poly(ethylene glycol) 2000 preferentially promoted the primer extensions with ATP and GTP by T7 RNA polymerase, regardless of Watson-Crick base-pairing rules. This indicates that molecular crowding decreases the dielectric constants in solution, resulting in enhancement of stacking interactions between the primer and an incorporated nucleotide. These findings suggest that molecular crowding could accelerate genetic diversity in the prebiotic world and may promote transcription error of RNA viruses in the current era. This journal is
DOI： 10.1039/d0ra06502a
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Effect of Molecular Crowding on the Stability of RNA G-Quadruplexes with Various Numbers of Quartets and Lengths of Loops. Reviewed
Saki Matsumoto, Hisae Tateishi-Karimata, Shuntaro Takahashi, Tatsuya Ohyama, Naoki Sugimoto
Biochemistry 59 ( 28 ) 2640 - 2649 2020.7