Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Murata Asako Last modified date:2024.06.03

Associate Professor / Department of Advanced Materials Science and Engineering / Faculty of Engineering Sciences


Papers
1. Qingwen Chen, Takeshi Yamada, Koichi Miyagawa, Asako Murata, Mitsuo Shoji, Kazuhiko Nakatani, A New Small Molecule DoNA Binding to CAG Repeat RNA, Bioorganic & Medicinal Chemistry, 10.1016/j.bmc.2023.117580, 98, 117580, 2023.12.
2. Qingwen Chen, Takeshi Yamada, Asako Murata, Ayako Sugai, Yasuyuki Matsushita, Kazuhiko Nakatani, A machine learning approach toward generating the focused molecule library targeting CAG repeat DNA, Digital Discovery, 10.1039/D3DD00160A, 3, 243-248, 2024.01,

This study evaluates a machine learning-based classification method with surface plasmon resonance (SPR) labeled data to create a focused molecule library. Our model increased the probability of hits from 5.2%...

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3. Yuzo Fujino, Morio Ueyama, Taro Ishiguro, Daisaku Ozawa, Hayato Ito, Toshihiko Sugiki, Asako Murata, Akira Ishiguro, Tania Gendron, Kohji Mori, Eiichi Tokuda, Tomoya Taminato, Takuya Konno, Akihide Koyama, Yuya Kawabe, Toshihide Takeuchi, Yoshiaki Furukawa, Toshimichi Fujiwara, Manabu Ikeda, Toshiki Mizuno, Hideki Mochizuki, Hidehiro Mizusawa, Keiji Wada, Kinya Ishikawa, Osamu Onodera, Kazuhiko Nakatani, Leonard Petrucelli, Hideki Taguchi, Yoshitaka Nagai, FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD., eLife, 10.7554/eLife.84338, 12, 2023.07, Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases..
4. Anisa Ulhusna, Asako Murata, Kazuhiko Nakatani, Inhibitory Effects of Mismatch Binding Molecules on the Repair Reaction of Uracil-Containing DNA, BIOCHEMISTRY, 10.1021/acs.biochem.2c00344, 61, 2522-2530, 2022.10, The stable R-loop formed during transcription induces enzyme-mediated deamination of cytosine, and the uracil in the DNA produced activates the base excision repair (BER) pathway. DNA cleavage involved in the BER pathway is thought to be one of the possible causes of trinucleotide repeat instability. Here, we performed an in vitro assay to investigate the effect of a DNA-binding small molecule, naphthyridine carbamate dimer (NCD), on BER enzyme reactions. The gel electrophoretic mobility shift assay (EMSA) and thermal melting analysis revealed the binding of NCD to a 5 '-XGG-3 '/5 '-XGG-3 ' triad (X = C or U or apurinic/ apyrimidinic site), which is a mimic of a BER enzyme substrate. Polyacrylamide gel electrophoresis (PAGE) of the reaction products of these substrates with hSMUG1 and APE1 enzymes in the presence of NCD showed that NCD interfered with the repair reaction in the 5 '-XGG-3 '/5 '-XGG-3 ' triad. These findings would broaden the potential of small molecules in modulating trinucleotide repeat instability..
5. Yusuke Takashima, Asako Murata, Kei Iida, Ayako Sugai, Masatoshi Hagiwara, Kazuhiko Nakatani, Method for Identifying Sequence Motifs in Pre-miRNAs for Small-Molecule Binding, ACS CHEMICAL BIOLOGY, 10.1021/acschembio.2c00452, 17, 10, 2817-2827, 2022.09, Non-coding RNAs are emerging targets for drug development because they are involved in various cellular processes. However, there are a few reliable design strategies for small molecules that can target RNAs. This paper reports a simple and efficient method to comprehensively analyze RNA motifs that can be bound by a specific small molecule. The method involves Dicer-mediated pre-miRNA cleavage and subsequent analysis of the reaction products by high-throughput sequencing. A pre-miRNA mutant library containing a randomized region at the Dicer deavage site was used as the substrate for the reaction. Sequencing analysis of the products of the reaction carried out in the presence or absence of a synthetic small molecule identified the pre-miRNA mutants whose Dicer-mediated deavage was significantly altered b y the addition of the small molecule. The binding of the small molecule to the identified pre-miRNA mutants was confirmed by surface plasmon resonance, demonstrating the feasibility of our method..
6. Hsiu-Ting Hsu, Asako Murata, Chikara Dohno, Kazuhiko Nakatani, KungYao Chang, Premature translation termination mediated non-ER stress induced ATF6 activation by a ligand-dependent ribosomal frameshifting circuit, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkac257, 50, 9, 5369-5383, 2022.05, The -1 programmed ribosomal frameshifting (-1 PRF) has been explored as a gene regulatory circuit for synthetic biology applications. The -1 PRF usually uses an RNA pseudoknot structure as the frameshifting stimulator. Finding a ligand-responsive pseudoknot with efficient -1 PRF activity is time consuming and is becoming a bottleneck for its development. Inserting a guanine to guanine (GG)-mismatch pair in the 5 '-stem of a small frameshifting pseudoknot could attenuate -1 PRF activity by reducing stem stability. Thus, a ligand-responsive frameshifting pseudoknot can be built using GG-mismatch-targeting small molecules to restore stem stability. Here, a pseudoknot requiring stem-loop tertiary interactions for potent frameshifting activity was used as the engineering template. This considerably amplified the effect of mismatch destabilization, and led to creation of a mammalian -1 PRF riboswitch module capable of mediating premature translation termination as a synthetic regulatory mode. Application of the synthetic circuit allowed ligand-dependent ATF6N mimic formation for the activation of protein folding-related genes involved in the unfolded protein response without an ER-stress inducing agent. With the availability of mismatch-targeting molecules, the tailored module thus paves the way for various mismatch plug-ins to streamline highly efficient orthogonal ligand-dependent -1 PRF stimulator development in the synthetic biology toolbox..
7. Lu Ni, Takeshi Yamada, Asako Murata, Kazuhiko Nakatani, Mismatch binding ligand upregulated back-splicing reaction producing circular RNA in a cellular model, CHEMICAL COMMUNICATIONS, 10.1039/d1cc06936e, 58, 22, 3629-3632, 2022.03, Circular RNA (circRNA) is a covalently closed single-stranded RNA produced from pre-mRNAs via back-splicing reaction, an alternative form of splicing. Here, we show naphthyridine carbamate dimer (NCD) upregulating the production of a circRNA from a pre-mRNA containing NCD-binding site UGGAA/UGGAA in cells, demonstrating the feasibility of small-molecule mediated circRNA production..
8. Sanjukta Mukherjee, Asako Murata, Ryoga Ishida, Ayako Sugai, Chikara Dohno, Michiaki Hamada, Sudhir Krishna, Kazuhiko Nakatani, HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules, MOLECULAR THERAPY-NUCLEIC ACIDS, 10.1016/j.omtn.2021.11.021, 27, 165-174, 2022.03, Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification prothe compound of interest and would contribute to increase the spectrum of biomedical application..
9. Bimolendu Das, Konami Nagano, Gota Kawai, Asako Murata, Kazuhiko Nakatani, 2-Amino-1,8-naphthyridine Dimer (ANP77), a High-Affinity Binder to the Internal Loops of C/CC and T/CC Sites in Double-Stranded DNA, JOURNAL OF ORGANIC CHEMISTRY, 10.1021/acs.joc.1c02383, 87, 1, 340-350, 2021.12, Small molecules targeting DNA regions with structural fluctuation are an important class of molecule as chemical probes for studying the role of these structures in biological systems and the development of neurological disorders. The molecule ANP77 we described here, where a three-atom linker connects two 2-amino-1,8-naphthyridines at the C7 position, was found to form stacked structure with protonation of naphthyridine at low pH, and bound to the internal loop consisting of C/CC and T/CC in double-stranded DNA with affinities of 4.8 and 34.4 nM, respectively. Mass spectrometry and isothermal titration calorimetry analyses determined the stoichiometry for the binding as 1:1, and chemical footprinting with permanganate and NMR structural analysis revealed that the T in the T/CC was forced to flip out toward an extrahelical position upon ANP77 binding. Protonated stacked ANP77 interacted with two adjacent cytosines through hydrogen bonding and occupied the position in the duplex by flipping out the C or T opposite CC. Finally, this study demonstrated the potential of ANP77 for binding to the sequences of biological significance with the TG(T/C)CC repeat of the PIG3 promoter and the telomere repeat CCCTAA..
10. Bimolendu Das, Asako Murata, Kazuhiko Nakatani, A small-molecule fluorescence probe ANP77 for sensing RNA internal loop of C, U and A/CC motifs and their binding molecules, Nucleic Acids Research, 10.1093/nar/gkab650, 49, 15, 8462-8470, 2021.09, Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam..
11. Tomoko Furuzono, Asako Murata, Satoshi Okuda, Kenji Mizutani, Tsuyoshi Adachi, Kazuhiko Nakatani, Speeding drug discovery targeting RNAs: An iterative “RNA selection-compounds screening cycle“ for exploring RNA-small molecule pairs, Bioorganic & Medicinal Chemistry, 10.1016/j.bmc.2021.116070, 36, 116070-116070, 2021.04, RNA is an emerging target of next-generation drug development. Recently, new small molecules targeting RNAs were discovered by several pharmaceutical companies. Methods have been reported to identify small molecules targeting a specific RNA sequence and structural motif, however, because of diverse sequence and structural motifs potentially present in the druggable functional RNAs, large sets of structure-activity relationships (SARs) information of small molecule - RNA interactions will be required for the acceleration and efficient startup of the discovery programs toward unprecedented RNA targets. Here we describe our iterative RNA selection and compounds screening to accumulate rich information about small molecules - RNA interaction. The RNAs that selectively bind to the initial molecular target, compound 1 from our in-house chemical library (JT-library), was isolated using in vitro selection technique from a hairpin-structured RNA library mimicking precursor microRNA (pre-miRNA). Then, we engineered pre-let-7f-2 to create its mutant that can bind to compound 1 by embedding the in vitro selected RNA motif for compound 1 in the hairpin loop region. The obtained mutant pre-let-7f-2-loop-mt was used as a target for screening 316 analogs of compound 1. A surface plasmon resonance (SPR) -based screening was performed against pre-let-7f-2-loop-mt-immobilized sensor surface and we obtained four compounds that can bind to the RNA. Among these four compounds, three compounds showed higher affinity to pre-let-7f-2-loop-mt than the parental compound 1, which suggests the feasibility of our strategy for gathering the SAR information on small molecule - RNA interactions. We demonstrated only one cycle of RNA selection and compounds screening in the present study, but can continue this cycle with the selected molecule to gain new RNAs and even new RNA motifs and gather much SAR information with improved accuracy..
12. Asako Murata, Yuki Mori, Yue Di, Ayako Sugai, Bimolendu Das, Yusuke Takashima, Kazuhiko Nakatani, Small Molecule-Induced Dimerization of Hairpin RNA Interfered with the Dicer Cleavage Reaction., Biochemistry, 10.1021/acs.biochem.0c00920, 60, 4, 245-249, 2021.02, MicroRNAs are potential targets for drug development. Small molecules that can inhibit or promote a specific miRNA's biogenesis would be useful for regulating its target genes. Various types of small molecules have been investigated so far for their potential application in modulating miRNA biogenesis. They bind to the target primary or precursor miRNAs and inhibit the processing of these precursors by Drosha or Dicer. However, the binding site that effectively interferes with the Dicer cleavage reaction is still undetermined. Here we report that our designed small molecule restricted naphthyridine dimer (RND) binds to the hairpin loop of a hairpin RNA and induces its dimerization. This study shows that the binding of the RND to the hairpin loop was not effective in interfering with the Dicer cleavage reaction, but dimerization of the hairpin RNA by RND binding effectively interfered with the Dicer cleavage reaction..
13. Kazuhiko Nakatani, Jun Matsumoto, Masayuki Nakamori, Tatsumasa Okamoto, Asako Murata, Chikara Dohno, The dimeric form of 1,3-diaminoisoquinoline derivative rescued the mis-splicing of Atp2a1 and Clcn1 genes in myotonic dystrophy type 1 mouse model., Chemistry (Weinheim an der Bergstrasse, Germany), 10.1002/chem.202001572, 26, 63, 14305-14309, 2020.05, Expanded CUG repeat RNA in the dystrophia myotonia protein kinase (DMPK) gene causes myotonic dystrophy type 1 (DM1) and sequesters RNA processing proteins, such as the splicing factor muscleblind-like 1 protein (MBNL1). Sequestration of splicing factors results in the mis-splicing of some pre-mRNAs. Small molecules that rescue the mis-splicing in the DM1 cells have drawn attention as potential drugs to treat DM1. Herein we report a new molecule JM642 consisted of two 1,3-diaminoisoquinoline chromophores having an auxiliary aromatic unit at the C5 position. JM642 alternates the splicing pattern of the pre-mRNA of the Ldb3 gene in the DM1 cell model and Clcn1 and Atp2a1 genes in the DM1 mouse model. In vitro binding analysis by surface plasmon resonance (SPR) assay to the r(CUG) repeat and disruption of ribonuclear foci in the DM1 cell model suggested the binding of JM642 to the expanded r(CUG) repeat in vivo, eventually rescue the mis-splicing..
14. Masayuki Nakamori, Gagan B Panigrahi, Stella Lanni, Terence Gall-Duncan, Hideki Hayakawa, Hana Tanaka, Jennifer Luo, Takahiro Otabe, Jinxing Li, Akihiro Sakata, Marie-Christine Caron, Niraj Joshi, Tanya Prasolava, Karen Chiang, Jean-Yves Masson, Marc S Wold, Xiaoxiao Wang, Marietta Y W T Lee, John Huddleston, Katherine M Munson, Scott Davidson, Mehdi Layeghifard, Lisa-Monique Edward, Richard Gallon, Mauro Santibanez-Koref, Asako Murata, Masanori P Takahashi, Evan E Eichler, Adam Shlien, Kazuhiko Nakatani, Hideki Mochizuki, Christopher E Pearson, A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo., Nature genetics, 10.1038/s41588-019-0575-8, 52, 2, 146-159, 2020.02, In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSβ. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats..
15. Takei Fumie, Akiyama Misaki, Murata Asako, Sugai Ayako, Nakatani Kazuhiko, Yamashita Ichiro, RT-Hpro-PCR: A MicroRNA Detection System Using a Primer with a DNA Tag, CHEMBIOCHEM, 10.1002/cbic.201900382, 21, 4, 477-480, 2019.11, MicroRNAs (miRNAs) are short RNAs that regulate the expression of complementary messenger RNAs and are involved in numerous human diseases. However, current detection techniques lack the sensitivity to detect miRNAs of low abundance. Moreover, at a length of 20-25 bases, miRNAs are too short for the reverse transcription (RT) polymerase chain reaction (PCR). Here we have developed a new, rapid, and simple miRNA detection system utilizing an RT primer containing a DNA tag at the 5'-end to increase the length of the cDNA. This strategy increases the length of the hybridized tagged primer and the complementary template DNA, as well as the melting temperature of the primer⋅template DNA duplex. PCR efficiency is thus increased, thereby enhancing miRNA detection sensitivity..
16. Mukherjee, S, Błaszczyk, L, Rypniewski, W, Falschlunger, C, Micura, R, Murata, A, Dohno, C, Nakatani, K, Kiliszek, A, Structural insights into synthetic ligands targeting A–A pairs in disease-related CAG RNA repeats, Nucl. Acids Res., 10.1093/nar/gkz832, 47, 20, 10906-10913, 2019.11, The trinucleotide repeat expansion disorders (TREDs) constitute of a group of >40 hereditary neurodegenerative human diseases associated with abnormal expansion of repeated sequences, such as CAG repeats. The pathogenic factor is a transcribed RNA or protein whose function in the cell is compromised. The disorders are progressive and incurable. Consequently, many ongoing studies are oriented at developing therapies. We have analyzed crystal structures of RNA containing CAG repeats in complex with synthetic cyclic mismatch-binding ligands (CMBLs). The models show well-defined interactions between the molecules in which the CMBLs mimic nucleobases as they form pseudo-canonical base pairs with adenosine residues and engage in extensive stacking interactions with neighboring nucleotides. The binding of ligands is associated with major structural changes of the CAG repeats, which is consistent with results of biochemical studies. The results constitute an early characterization of the first lead compounds in the search for therapy against TREDs. The crystallographic data indicate how the compounds could be further refined in future biomedical studies..
17. Murata Asako, Nakamori Masayuki, Nakatani Kazuhiko, Modulating RNA secondary and tertiary structures by mismatch binding ligands, METHODS, 10.1016/j.ymeth.2019.05.006, 167, 78-91, 2019.09, Much recent attention has been focused on small organic molecules binding to non-canonical structures of nucleic acids, especially, RNA. The Human Genome Project and the ENCODE (encyclopedia of DNA elements) project revealed that more than 75% of the human genome is transcribed into RNA, while only ∼3% of the human genome encodes a protein. These non-protein-coding RNAs are thought to play significant roles in many cellular processes and are promising targets for drug discovery. Emerging roles of the non-coding RNAs in a variety of diseases provides enormous opportunities for pharmaceutical research on developing drugs targeting undruggable and rare diseases. During the last two decades, our laboratory has focused attention on small molecules binding to non-canonical DNA and RNA structures, especially to mismatched base pairs. Mismatch binding ligands (MBLs) we have developed are synthetic molecules designed in silico based on the hypothesis of hydrogen-bonding and semi-intercalation to DNA and RNA. Most of MBLs consists of two heterocycles having hydrogen bonding surfaces fully or partially complementary to that of nucleotide bases. In our design, each heterocycle binds to one of the mismatched bases by hydrogen bonding to form pseudo-base pairs, which would be stacked with the adjacent base pairs. The hypothesis allows us in principle to design small molecules binding to any mismatched base pairs, but it turned out not to be the case in reality. However, we have so far succeeded in developing several MBLs binding to DNA and RNA motifs of biological significance. In this review, we shall describe the hypothesis of molecular design of MBLs and its outcome regarding RNA targeting..
18. Otabe Takahiro, Nagano Konami, Kawai Gota, Murata Asako, Nakatani Kazuhiko, Inhibition of pre-miRNA-136 processing by Dicer with small molecule BzDANP suggested the formation of ternary complex of pre-miR-136-BzDANP-Dicer, BIOORGANIC & MEDICINAL CHEMISTRY, 10.1016/j.bmc.2019.03.031, 27, 10, 2140-2148, 2019.05, Small-molecule modulators, along with antisense oligonucleotide, would be powerful tools and potential drug candidates for modulating miRNA-related gene expressions. The mechanism of the inhibitory effect of the C-bulge binding small molecule BzDANP for the Dicer processing reaction of pre-miR-136 was discussed on the data obtained by SPR, NMR, and kinetic analysis for Dicer processing. SPR and NMR analysis showed the preference of BzDANP binding to the C-bulge. Michaelis-Menten analysis suggested the formation of a ternary complex pre-miR-136-BzDANP-Dicer during the Dicer-cleavage reaction of pre-miR-136 in the presence of BzDANP. The inhibitory effect of BzDANP is likely attributed to the slower reaction from the ternary complex than that from the binary pre-miR-136-Dicer complex.
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19. Li Jinxing, Nakamori Masayuki, Matsumoto Jun, Murata Asako, Dohno Chikara, Kiliszek Agnieszka, Taylor Katarzyna, Sobczak Krzysztof, Nakatani Kazuhiko, A Dimeric 2,9-Diamino-1,10-phenanthroline Derivative Improves Alternative Splicing in Myotonic Dystrophy Type 1 Cell and Mouse Models, CHEMISTRY-A EUROPEAN JOURNAL, 10.1002/chem.201804368, 24, 68, 18115-18122, 2018.12, Expanded r(CUG) repeats are the cause of the neurological disorder myotonic dystrophy type 1 (DM1). The pathological features of DM1 include the formation of ribonuclear foci containing expanded r(CUG) repeats, which sequester the MBNL1 protein and lead to the misregulation of alternative pre-mRNA splicing. Small molecules that bind to the r(CUG) repeats and improve alternative splicing have therapeutic potential in the treatment of DM1. Herein, the synthesis of DDAP (a dimeric form of the CUG-binding molecule DAP reported previously), its binding properties to r(CUG) repeats, and its effect on the misregulation of splicing are reported. The surface plasmon resonance assay, circular dichroism spectra, and ESI-TOF mass spectrometry results confirmed the binding of DDAP to r(CUG)9 repeats. Studies on a DM1 cell model and a DM1 mouse model revealed that DDAP was partially effective in the recovery of the pre-mRNA splicing defects. The mechanism underlying this recovery was studied in vitro through a competitive binding assay, and suggested that DDAP could interfere with the binding of MBNL1 to r(CUG) repeats in a concentration-dependent manner.
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20. Matsumoto Saki, Caliskan Neva, Rodnina Marina V, Murata Asako, Nakatani Kazuhiko, Small synthetic molecule-stabilized RNA pseudoknot as an activator for-1 ribosomal frameshifting, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gky689, 46, 16, 8079-8089, 2018.09, Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible -1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.
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21. Kazuhiko Nakatani, Nozomi Natsuhara, Yuki Mori, Sanjukta Mukherjee, Bimolendu Das, Asako Murata, Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA, CHEMISTRY-AN ASIAN JOURNAL, 10.1002/asia.201701293, 12, 23, 3077-3087, 2017.12, One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small-molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1-NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1-NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1-NHR was performed from pre-miR-29a loop library RNA, and one RNA, to which 1-NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1-NHR, with a predominantly unstacked conformation, would show entropy-driven binding, isothermal titration calorimetry analysis suggested that the binding of 1-NHR to RNA was enthalpy driven with an apparent K-d of about 100nm..
22. Saki Matsumoto, Kei Iida, Asako Murata, Masatsugu Denawa, Masatoshi Hagiwara, Kazuhiko Nakatani, Synthetic ligand promotes gene expression by affecting GC sequence in promoter, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 10.1016/j.bmcl.2017.06.006, 27, 15, 3391-3394, 2017.08, A naphthyridine carbamate tetramer (NCT8) is a synthetic compound, which selectively binds to nucleic acids containing CGG/CGG sequence. Although NCT8 is a promising compound for a wide range of DNA and RNA based biotechnology such as modulation of specific gene expression, little is known about its behavior in human cells. In the present study, we investigated the changes induced in gene expression by NCT8. Genes differentially expressed in the presence of NCT8 in HeLa cells were identified by whole-transcriptome analysis. The whole-transcriptome analysis showed that NCT8 significantly induced up-regulation of specific genes, whose promoter region has GC-rich sequence. (C) 2017 Elsevier Ltd. All rights reserved..
23. Jinxing Li, Jun Matsumoto, Li-Ping Bai, Asako Murata, Chikara Dohno, Kazuhiko Nakatani, A Ligand That Targets CUG Trinucleotide Repeats, CHEMISTRY-A EUROPEAN JOURNAL, 10.1002/chem.201602741, 22, 42, 14881-14889, 2016.10, The development of small molecules that can recognize specific RNA secondary and tertiary structures is currently an important research topic for developing tools to modulate gene expression and therapeutic drugs. Expanded CUG trinucleotide repeats, known as toxic RNA, capture the splicing factor MBNL1 and are causative of neurological disorder myotonic dystrophy type 1 (DM1). Herein, the rational molecular design, synthesis, and binding analysis of 2,9-diaminoalkyl-substituted 1,10-phenanthroline (DAP), which bound to CUG trinucleotide repeats, is described. The results of melting temperature (T-m) analyses, surface plasmon resonance (SPR) assay, and electrospray spray ionization time-of-flight (ESI-TOF) mass spectrometry showed that DAP bound to r(CUG)(9) but not to r(CAG)(9) and r(CGG)(9). The dual luciferase assay clearly indicated DAP bound to the r(CUG)(n) repeat by affecting the translation in vitro..
24. Asako Murata, Takahiro Otabe, Jinhua Zhang, Kazuhiko Nakatani, BzDANP, a Small-Molecule Modulator of Pre-miR-29a Maturation by Dicer, ACS CHEMICAL BIOLOGY, 10.1021/acschembio.6b00214, 11, 10, 2790-2796, 2016.10, We here report the synthesis of novel molecule BzDANP having a three-ring benzo[c][1,8]naphthyridine system, the evaluation of its binding properties to a single nucleotide bulge in RNA duplexes, and BzDANP-induced suppression of pre-miR-29a processing by Dicer. BzDANP showed much increased affinity to the bulged RNAs as compared with the parent molecule DANP, which possesses the same hydrogen-bonding surface as BzDANP but in a two-ring [1,8]naphthyridine system. Melting temperature analysis of bulged RNAs showed that BzDANP most effectively stabilized the C-bulged RNA. Dicer-mediated processing of pre-miR-29a was suppressed by BzDANP in a concentration dependent manner. The presence of the C-bulge at the Dicer cleavage site was effective for the suppression of pre-miR-29a processing by BzDANP. These results demonstrated that the small molecule binding to the bulged site in the vicinity of the Dicer cleavage site could be a potential modulator for the maturation of pre-miRNA..
25. Jinxing Li, Akihiro Sakata, Hanping He, Li-Ping Bai, Asako Murata, Chikara Dohno, Kazuhiko Nakatani, Naphthyridine-Benzoazaquinolone: Evaluation of a Tricyclic System for the Binding to (CAG)(n) Repeat DNA and RNA, CHEMISTRY-AN ASIAN JOURNAL, 10.1002/asia.201600527, 11, 13, 1971-1981, 2016.07, The expansion of CAG repeats in the human genome causes the neurological disorder Huntington's disease. The small-molecule naphthyridine-azaquinolone NA we reported earlier bound to the CAG/CAG motif in the hairpin structure of the CAG repeat DNA. In order to investigate and improve NA-binding to the CAG repeat DNA and RNA, we conducted systematic structure-binding studies of NA to CAG repeats. Among the five new NA derivatives we synthesized, surface plasmon resonance (SPR) assay showed that all of the derivatives modified from amide linkages in NA to a carbamate linkage failed to bind to CAG repeat DNA and RNA. One derivative, NBzA, modified by incorporating an additional ring to the azaquinolone was found to bind to both d(CAG)(9) and r(CAG)(9). NBzA binding to d(CAG)(9) was similar to NA binding in terms of large changes in the SPR assay and circular dichroism (CD) as well as pairwise binding, as assessed by electron spray ionization time-of-flight (ESI-TOF) mass spectrometry. For the binding to r(CAG)(9), both NA and NBzA showed stepwise binding in ESI-TOF MS, and NBzA-binding to r(CAG)(9) induced more extensive conformational change than NA-binding. The tricyclic system in NBzA did not show significant effects on the binding, selectivity, and translation, but provides a large chemical space for further modification to gain higher affinity and selectivity. These studies revealed that the linker structure in NA and NBzA was suitable for the binding to CAG DNA and RNA, and that the tricyclic benzoazaquinolone did not interfere with the binding..
26. Takeo Fukuzumi, Asako Murata, Haruo Aikawa, Yasue Harada, Kazuhiko Nakatani, Exploratory Study on the RNA-Binding Structural Motifs by Library Screening Targeting pre-miRNA-29a, CHEMISTRY-A EUROPEAN JOURNAL, 10.1002/chem.201502913, 21, 47, 16859-16867, 2015.11, The metabolic stream of microRNA (miRNA) production, the so-called maturation process of miRNAs, became one of important metabolic paths for drug-targeting to modulate the expression of genes related to a number of diseases. We carried out discovery studies on small molecules binding to the precursor of miR-29a (pre-miR-29a) from a chemical library containing 41119 compounds (AQ library) by the fluorescent indicator displacement (FID) assay using the xanthone derivative X2SdiMe as a fluorescent indicator. The FID assay provided 1075 compounds, which showed an increase of fluorescence. These compounds were subsequently submitted to a binding analysis in a surface plasmon resonance (SPR) assay on a pre-miR-29a immobilized surface. 21 hit compounds were identified with a good reproducibility in the binding. These compounds have not been reported to bind to RNA until now and can be classified into two groups on the basis of the kinetics in the binding. To gain more information on the motif structures that could be necessary for the binding to pre-miR-29a, 19 sub-structures were selected from the hit compounds. The substructure library (SS library) which consisted of 362 compounds was prepared from the AQ library. An SPR assay of the SS library on pre-miR-29a-immobilized surface suggested that five substructures could potentially be important structural motifs to bind to pre-miR-29a. These studies demonstrate that the combination of FID-based screening of chemical library and subsequent SPR assay would be one way for obtaining practical solutions for the discovery of molecules which bind to the target pre-miRNAs..
27. Shin-ichi Sato, Mizuki Watanabe, Yousuke Katsuda, Asako Murata, Dan Ohtan Wang, Motonari Uesugi, Live-Cell Imaging of Endogenous mRNAs with a Small Molecule, ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 10.1002/anie.201410339, 54, 6, 1855-1858, 2015.02, Determination of subcellular localization and dynamics of mRNA is increasingly important to understanding gene expression. A new convenient and versatile method is reported that permits spatiotemporal imaging of specific non-engineered RNAs in living cells. The method uses transfection of a plasmid encoding a gene-specific RNA aptamer, combined with a cell-permeable synthetic small molecule, the fluorescence of which is restored only when the RNA aptamer hybridizes with its cognitive mRNA. The method was validated by live-cell imaging of the endogenous mRNA of -actin. Application of the technology to mRNAs of a total of 84 human cytoskeletal genes allowed us to observe cellular dynamics of several endogenous mRNAs including arfaptin-2, cortactin, and cytoplasmic FMR1-interacting protein2. The RNA-imaging technology and its further optimization might permit live-cell imaging of any RNA molecules..
28. Takeo Fukuzumi, Haruo Aikawa, Yasue Harada, Ayako Sugai, Asako Murata, Kazuhiko Nakatani, Synthesis of 8-Substituted Adenine and Adenosine Libraries and the Binding to pre-miR-29a, BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN, 10.1246/bcsj.20140137, 87, 9, 1013-1015, 2014.09, A small chemical library of 8-substituted adenine and adenosine derivatives was synthesized and examined for binding to pre-miR-29a. We found that one compound showed the affinity with 61 nM of K-d and a 10-fold stronger binding than the double-stranded RNA..
29. Changfeng Hong, Takahiro Otabe, Saki Matsumoto, Chikara Dohno, Asako Murata, Masaki Hagihara, Kazuhiko Nakatani, Formation of a Ligand- Assisted Complex of Two RNA Hairpin Loops, CHEMISTRY-A EUROPEAN JOURNAL, 10.1002/chem.201304683, 20, 18, 5282-5287, 2014.04, The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)(3) in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Forster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs..
30. Nao Hirata, Masato Nakagawa, Yuto Fujibayashi, Kaori Yamauchi, Asako Murata, Itsunari Minami, Maiko Tomioka, Takayuki Kondo, Ting-Fang Kuo, Hiroshi Endo, Haruhisa Inoue, Shin-ichi Sato, Shin Ando, Yoshinori Kawazoe, Kazuhiro Aiba, Koh Nagata, Eihachiro Kawase, Young-Tae Chang, Hirofumi Suemori, Koji Eto, Hiromitsu Nakauchi, Shinya Yamanaka, Norio Nakatsuji, Kazumitsu Ueda, Motonari Uesugi, A Chemical Probe that Labels Human Pluripotent Stem Cells, CELL REPORTS, 10.1016/j.celrep.2014.02.006, 6, 6, 1165-1174, 2014.03, A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents..
31. Asako Murata, Shin-Ichi Sato, In vitro selection of RNA aptamers for a small-molecule dye, Methods in Molecular Biology, 10.1007/978-1-62703-755-6_2, 1111, 17-28, 2014.01, Artificial riboswitches can be generated by functional coupling of an RNA aptamer for synthetic small molecule to an expression platform. RNA aptamers that can bind strongly and selectively to their target are feasible to be used for obtaining more potent artificial riboswitches. In this chapter, we describe tips and notes for in vitro selection of RNA aptamers targeting synthetic small molecules. © Springer Science+Business Media New York 2014..
32. Asako Murata, Yasue Harada, Takeo Fukuzumi, Kazuhiko Nakatani, Fluorescent indicator displacement assay of ligands targeting 10 microRNA precursors, BIOORGANIC & MEDICINAL CHEMISTRY, 10.1016/j.bmc.2013.09.007, 21, 22, 7101-7106, 2013.11, Fluorescent indicator displacement (FID) assay is a rapid and convenient assay for identifying new ligands that bind to the target molecules. In our previous studies, we have shown that a series of 2,7-diaminoalkoxy xanthone and thioxanthone derivatives can be used as fluorescent indicators for detecting the interaction between RNA and a ligand. The xanthone and thioxanthone fluorochromes showed efficient fluorescence quenching upon binding to target RNA. Subsequent displacement of the bound-fluorochrome with a ligand that binds more strongly to the target RNA led to the recovery of the fluorescence by releasing the fluorochrome from RNA. Here we report a pilot screening of a chemical library that contains 9600 structurally diverse compounds for molecules that bind to a specific miRNA precursor using the FID assay. (C) 2013 Elsevier Ltd. All rights reserved..
33. Saki Matsumoto, Changfeng Hong, Takahiro Otabe, Asako Murata, Kazuhiko Nakatani, Ligand-inducible formation of RNA pseudoknot, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 10.1016/j.bmcl.2013.04.037, 23, 12, 3539-3541, 2013.06, Here, we demonstrate that a series of naphthyridine derivatives, naphthyridine carbamate tetramer (NCTn), can bind to the RNA CGG/CGG triad comprised of two single-stranded regions of a hairpin loop and a tail. Complete suppression of the binding by a single mutation indicated simultaneous binding of NCTn between hairpin loop and single stranded tail, leading to the formation of NCTn-induced pseudoknot. (C) 2013 Elsevier Ltd. All rights reserved..
34. Asako Murata, Takeo Fukuzumi, Shiori Umemoto, Kazuhiko Nakatani, Xanthone derivatives as potential inhibitors of miRNA processing by human Dicer: Targeting secondary structures of pre-miRNA by small molecules, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 10.1016/j.bmcl.2012.10.108, 23, 1, 252-255, 2013.01, In recent years, various biological processes have been found to be regulated by miRNA-mediated gene silencing. A small molecule that modulate the miRNA pathway will provide the biological tool for elucidating mechanisms of miRNA-mediated gene regulation, and can be the drug lead for miRNA related diseases. In this study, we demonstrated that an aminoalkoxy-substituted thioxanthone derivative interferes Dicer-mediated processing of pre-miRNA. Information about the interaction between these xanthone derivatives and pre-miRNAs will enable us to design and develop new small molecule-based inhibitors for miRNA pathway. (c) 2012 Published by Elsevier Ltd..
35. Shiori Umemoto, Seongwang Im, Jinhua Zhang, Masaki Hagihara, Asako Murata, Yasue Harada, Takeo Fukuzumi, Takahiro Wazaki, Shin-ichi Sasaoka, Kazuhiko Nakatani, Structure-Activity Studies on the Fluorescent Indicator in a Displacement Assay for the Screening of Small Molecules Binding to RNA, CHEMISTRY-A EUROPEAN JOURNAL, 10.1002/chem.201103932, 18, 32, 9999-10008, 2012.08, A series of xanthone and thioxanthone derivatives with aminoalkoxy substituents were synthesized as fluorescent indicators for a displacement assay in the study of small-moleculeRNA interactions. The RNA-binding properties of these molecules were investigated in terms of the improved binding selectivity to the loop region in the RNA secondary structure relative to 2,7-bis(2-aminoethoxy)xanthone (X2S) by fluorimetric titration and displacement assay. An 11-mer double-stranded RNA and a hairpin RNA mimicking the stem loop IIB of Rev response element (RRE) RNA of HIV-1 mRNA were used. The X2S derivatives with longer aminoalkyl substituents showed a higher affinity to the double-stranded RNA than the parent molecule. Introduction of a methyl group on the aminoethoxy moiety of X2S effectively modulated the selectivity to the RNA secondary structure. Methyl group substitution at the C1' position suppressed the binding to the loop regions. Substitution with two methyl groups on the amino nitrogen atom resulted in reducing the affinity to the double-stranded region by a factor of 40?%. The effect of methyl substitution on the amino nitrogen atom was also observed for a thioxanthone derivative. Titration experiments, however, suggested that thioxanthone derivatives showed a more prominent tendency of multiple binding to RNA than xanthone derivatives. The selectivity index calculated from the affinity to the double-stranded and loop regions suggested that the N,N-dimethyl derivative of X2S would be suitable for the screening of small molecules binding to RRE..
36. Asako Murata, Shin-ichi Sato, Yoshinori Kawazoe, Motonari Uesugi, Small-molecule fluorescent probes for specific RNA targets, Chemical Communications, 10.1039/c1cc10393h, 47, 16, 4712-4712, 2011.05, A method was developed that uses small molecules as fluorescent probes to detect specific mRNAs. In this approach, the fluorescence of fluorophore–quencher conjugates is restored by the binding of an mRNA aptamer tag to the quencher segment of the molecules. The method allows real-time detection of mRNA transcripts in vitro..
37. Shin-ichi Sato, Asako Murata, Tsubasa Orihara, Takashi Shirakawa, Kiyotake Suenaga, Hideo Kigoshi, Motonari Uesugi, Marine Natural Product Aurilide Activates the OPA1-Mediated Apoptosis by Binding to Prohibitin, CHEMISTRY & BIOLOGY, 10.1016/j.chembiol.2010.10.017, 18, 1, 131-139, 2011.01, Aurilide is a potent cytotoxic marine natural product that induces apoptosis in cultured human cells at the picomolar to nanomolar range; however, its mechanism of action has been unknown. Results of the present study showed that aurilide selectively binds to prohibitin 1 (PHB1) in the mitochondria, activating the proteolytic processing of optic atrophy 1 (OPA1) and resulting in mitochondria-induced apoptosis. The mechanism of aurilide cytotoxicity suggests that PHB1 is an apoptosis-regulating protein amenable to modulation by small molecules. Aurilide may serve as a small-molecule tool for studies of mitochondria-induced apoptosis..
38. Shin-ichi Sato, Asako Murata, Takashi Shirakawa, Motonari Uesugi, Biochemical Target Isolation for Novices: Affinity-Based Strategies, CHEMISTRY & BIOLOGY, 10.1016/j.chembiol.2010.05.015, 17, 6, 616-623, 2010.06, Although a number of genomic and biochemical technologies are now used to elucidate the mechanisms of action of bioactive small molecules, affinity-based isolation of molecular targets is a classic, but still powerful, approach. This review highlights recent cases where biochemical isolation of target proteins of bioactive small molecules highlighted general strategies for a successful isolation and identification of molecular targets. This review is intended to be both an update on the most recent findings for those already active in the field of forward chemical genetics and a guide for scientists entering this burgeoning field..
39. Asako Murata, Takeshi Wada, Synthesis of a novel ester analog of nucleic acids bearing a serine backbone, Bioorganic and Medicinal Chemistry Letters, 10.1016/j.bmcl.2006.02.076, 16, 11, 2933-2936, 2006.06, A novel analog of nucleic acids bearing an optically active serine ester backbone, serine-based nucleobase-linked polyester (SNE), was synthesized. Monomers containing a thymine base were synthesized from l- and d-serines. Furthermore, reaction conditions were thoroughly examined for the ester bond formation by using a new phosphonium-type condensing reagent on a solid support without racemization. The release of the dimer from the resin was also investigated using a new type of linker, which could be cleaved under neutral conditions. © 2006 Elsevier Ltd. All rights reserved..
40. Asako Murata, Takeshi Wada, A novel linker for solid-phase synthesis cleavable under neutral conditions, Tetrahedron Letters, 10.1016/j.tetlet.2006.01.135, 47, 13, 2147-2150, 2006.03, A novel linker cleavable under neutral conditions has been developed for the solid-phase synthesis of base-labile compounds. The linker is comprised of a 3-azidomethyl-4-hydroxybenzyl alcohol moiety, and the azidomethyl group in the linker is readily converted to an aminomethyl group by treatment with a phosphine reagent in the presence of water to result in an intramolecular cyclization to release the compounds. Using the linker, a base-labile dinucleoside methyl phosphate was synthesized on a highly cross-linked polystyrene (HCP) support and cleaved successfully from the resin without decomposition of the product. © 2006 Elsevier Ltd. All rights reserved..