論文 - 杉本 直己
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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
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S. Takahashi, S. Bhowmik, S. Sato, S. Takenaka, and N. Sugimoto
Life 12 ( 4 ) 553 2022年4月
担当区分：最終著者
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 査読あり 国際共著 国際誌
Kane T. McQuaid, Shuntaro Takahashi, Lena Baumgaertner, David J. Cardin, Neil G. Paterson, James P. Hall, Naoki Sugimoto, and Christine J. Cardin
Journal of the American Chemical Society 144 ( 13 ) 5956 - 5964 2022年4月
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.
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細胞内での核酸の挙動を明らかにする熱力学的解析 査読あり 国際誌
高橋俊太郎, 杉本 直己
熱測定 49 14 - 19 2022年1月
共著
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S. Takahashi, S. Matsumoto, P. Chilka, S. Ghosh, H. Okura, and N. Sugimoto
Scientific reports 12 1149 2022年1月
担当区分：最終著者
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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S.Matsumoto, H. Tateishi-Karimata, T. Ohyama, and N. Sugimoto
RSC Advances 11 37205 - 37217 2021年12月
共著
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W. Sugimoto, N. Kinoshita, M. Nakata, T. Ohyama, H. Tateishi-Karimata, T. Nishikata, N. Sugimoto, D. Miyoshi, and K. Kawauchi
Chem. Commun. 58 48 - 51 2021年11月
共著
DOI： 10.1039/d1cc05523b
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Dipanwita Banerjee, Hisae Tateishi-Karimata, Tatsuya Ohyama, Saptarshi Ghosh, Tamaki Endoh, Shuntaro Takahashi, Naoki Sugimoto
Nucleic acids research 49 ( 18 ) 10796 - 10799 2021年10月
The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.
DOI： 10.1093/nar/gkab780
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Chemical Modulation of DNA Replication along G-Quadruplex Based on Topology-Dependent Ligand Binding 査読あり 国際誌
高橋 俊太郎, 建石 寿枝, 杉本 直己
Journal of the American Chemical Society 143 16458 - 16469 2021年9月
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S.Satopathi, T.Endoh, Peter Podbevˇsek, Janez Plavec, N. Sugimoto
Nucleic acids research 49 8449 - 8461 2021年9月
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Triple-Helical Binding of Peptide Nucleic Acid Inhibits Maturation of Endogenous MicroRNA-197 査読あり 国際共著 国際誌
遠藤 玉樹, 杉本 直己
ACS Chemical biology 16 1147 - 1151 2021年7月
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N. Sugimoto, T. Endoh, S. Takahashi, H. Tateishi-Karimata
Bulletin of the Chemical Society of Japan 94 1970 - 1998 2021年7月
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Enhancement of the Catalytic Activity of Hammerhead Ribozymes by Organic Cations. 査読あり 国際誌
Shu-Ichi Nakano, Hirofumi Yamashita, Naoki Sugimoto
Chembiochem : a European journal of chemical biology 2021年7月
Catalytic turnover is important for the application of ribozymes to biotechnology. However, the turnover is often impaired because of the intrinsic high stability of base pairs with cleaved RNA products. Here, organic cations were used as additives to improve the catalytic performance of hammerhead ribozyme constructs that exhibit different kinetic behaviors. Kinetic analysis of substrate cleavage demonstrated that bulky cations, specifically tetra-substituted ammonium ions containing pentyl groups or a benzyl group, have the ability to greatly increase the turnover rate of the ribozymes. Thermal stability analysis of RNA structures revealed that the bulky cations promote the dissociation of cleaved products and refolding of incorrectly folded structures with small disruption of the catalytic structure. The use of bulky cations is a convenient method for enhancing the catalytic activity of hammerhead ribozymes, and the approach may be useful for advancing ribozyme technologies.
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New Insights into the Functions of Nucleic Acids Controlled by Cellular Microenvironments Topics in Current Chemistry 査読あり 国際誌
S.Matsumoto, N.Sugimoto
Topics in Current Chemistry 379 2021年6月
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Artificial turn-on riboswitch to control target gene expression using a wild-type riboswitch splicing mechanism 査読あり 国際誌
T. Yamauchi, T. Kubodera, D. Miyoshi, N. Sugimoto, and S. Hirohata
Journal of Bioscience and Bioengineering 131 115 - 123 2021年2月
共著
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M. Tsuruta, Y. Sugitani, N. Sugimoto, D. Miyoshi
Int. J. Mol. Sci., 22 947 2021年1月
共著
DOI： 10.3390/ijms22020947
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Engineering exosome polymer hybrids by atom transfer radical polymerization 査読あり 国際共著 国際誌
S. Lathwal, S. S. Yerneni, S. Boye, U. L. Muza, S. Takahashi, N. Sugimoto, A. Lederer, S. R. Das, P. G. Campbell, K. Matyjaszewski
Proc. Natl. Acad. Sci. U. S. A., 118 e2020241118 2021年1月
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