| 1. |
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.. |
| 2. |
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.. |
| 3. |
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.. |
| 4. |
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.. |
| 5. |
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.. |
| 6. |
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.. |
| 7. |
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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| 8. |
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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| 9. |
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.. |
| 10. |
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.. |
| 11. |
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.. |
| 12. |
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.. |
| 13. |
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.. |
| 14. |
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.. |
| 15. |
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.. |
| 16. |
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.. |
