Papers - ENDOH Tamaki
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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. Invited Reviewed International journal
Tamaki Endoh, Shuntaro Takahashi, Naoki Sugimoto
Chemical communications (Cambridge, England) 59 ( 7 ) 872 - 875 2023.1
Authorship：Lead author
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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Cladogenetic Orthogonal Light-Up Aptamers for Simultaneous Detection of Multiple Small Molecules in Cells. Reviewed International coauthorship International journal
Tamaki Endoh, Jia-Heng Tan, Shuo-Bin Chen, Naoki Sugimoto
Analytical chemistry 95 ( 2 ) 976 - 985 2023.1
Authorship：Lead author, Corresponding author
Recent successes in construction of light-up RNA aptamers allowed fluorescence-based live-cell imaging of RNAs. In addition, light-up aptamers have been converted into signaling aptamers that enable fluorometric detection of small chemicals. To date, only a single target chemical has been detected at a time in cells. In this study, we selected cladogenetic orthogonal light-up aptamers that output three different colors from the RNA library having the same ligand binding core. Two of the three functioned in mammalian cells. These two aptamers, which fluoresce blue and green upon binding of cognate fluorogen, were converted into signaling aptamers. Using these signaling aptamers in combination with a previously described light-up aptamer with red fluorescence, we demonstrated simultaneous detection of multiple chemicals in living cells. The cladogenetic orthogonal light-up aptamers developed in this study and the simple strategy for rational designing of the signaling aptamers will provide innovative advances in the field of RNA-based bioimaging.
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High-temperature adaptation of an OsNRT2.3 allele is thermoregulated by small RNAs. Reviewed International coauthorship International journal
Yong Zhang, Hisae Tateishi-Karimata, Tamaki Endoh, Qiongli Jin, Kexin Li, Xiaoru Fan, Yingjun Ma, Limin Gao, Haiyan Lu, Zhiye Wang, Art E Cho, Xuefeng Yao, Chunming Liu, Naoki Sugimoto, Shiwei Guo, Xiangdong Fu, Qirong Shen, Guohua Xu, Luis Rafael Herrera-Estrella, Xiaorong Fan
Science advances 8 ( 47 ) eadc9785 2022.11
Climate change negatively affects crop yield, which hinders efforts to reach agricultural sustainability and food security. Here, we show that a previously unidentified allele of the nitrate transporter gene OsNRT2.3 is required to maintain high yield and high nitrogen use efficiency under high temperatures. We demonstrate that this tolerance to high temperatures in rice accessions harboring the HTNE-2 (high temperature resistant and nitrogen efficient-2) alleles from enhanced translation of the OsNRT2.3b mRNA isoform and the decreased abundance of a unique small RNA (sNRT2.3-1) derived from the 5' untranslated region of OsNRT2.3. sNRT2.3-1 binds to the OsNRT2.3a mRNA in a temperature-dependent manner. Our findings reveal that allelic variation in the 5' untranslated region of OsNRT2.3 leads to an increase in OsNRT2.3b protein levels and higher yield during high-temperature stress. Our results also provide a breeding strategy to produce rice varieties with higher grain yield and lower N fertilizer input suitable for a sustainable agriculture that is resilient against climate change.
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Applicability of the nearest-neighbour model for pseudoknot RNAs. Reviewed International journal
Sagar Satpathi, Tamaki Endoh, Naoki Sugimoto
Chemical Communications 58 ( 40 ) 5952 - 5955 2022.5
The validity of the nearest-neighbour (NN) model was verified in an RNA pseudoknot (PK) structure. The thermodynamic parameters of the second hairpin stem (S2) region, which separates the PK from a hairpin structure, were monitored using CD and UV melting. Different PKs with identical NN base pairs in the S2 region exhibited similar thermodynamic parameters, highlighting the validity of the NN model in this RNA tertiary structure motif.
DOI： 10.1039/d1cc07094k
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Correction to 'Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition'. 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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Transcriptome screening followed by integrated physicochemical and structural analyses for investigating RNA-mediated berberine activity. Reviewed International coauthorship International journal
Sagar Satpathi, Tamaki Endoh, Peter Podbevšek, Janez Plavec, Naoki Sugimoto
Nucleic acids research 49 ( 15 ) 8449 - 8461 2021.9
Authorship：Lead author
Non-coding RNAs are regarded as promising targets for the discovery of innovative drugs due to their abundance in the genome and their involvement in many biological processes. Phytochemicals (PCs) are the primary source of ligand-based drugs due to their broad spectrum of biological activities. Since many PCs are heterocyclic and have chemical groups potentially involved in the interaction with nucleic acids, detailed interaction analysis between PCs and RNA is crucial to explore the effect of PCs on RNA functions. In this study, an integrated approach for investigating interactions between PCs and RNAs were demonstrated to verify the RNA-mediated PCs functions by using berberine (BRB) as a model PC. RNA screening of a transcriptome library followed by sequence refinement found minimal RNA motif consisting of a cytosine bulge with U-A and G-U neighbouring base pairs for interaction with BRB. NMR-based structure determination and physicochemical analyses using chemical analogues of BRB demonstrated the importance of electrostatic and stacking interactions for sequence selective interaction and RNA stabilization. The selective interaction with a relatively small RNA motif based on a chemical structure of a planer heterocyclic highlights the biological activities of various PCs mediated by the interactions with particular functional RNAs. In addition, the systematic and quantitative investigations demonstrated in this study could be useful for the development of therapeutic chemicals targeting functional RNAs, based on the PCs, in the future.
DOI： 10.1093/nar/gkab189
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Triple-Helical Binding of Peptide Nucleic Acid Inhibits Maturation of Endogenous MicroRNA-197. Reviewed International coauthorship International journal
Tamaki Endoh, Nikita Brodyagin, Dziyana Hnedzko, Naoki Sugimoto, Eriks Rozners
ACS chemical biology 16 ( 7 ) 1147 - 1151 2021.7
Authorship：Lead author
Sequence specific recognition and functional inhibition of biomedically relevant double-helical RNAs is highly desirable but remains a formidable problem. The present study demonstrates that electroporation of a triplex-forming peptide nucleic acid (PNA), modified with 2-aminopyridine (M) nucleobases, inhibited maturation of endogenous microRNA-197 in SH-SY5Y cells, while having little effect on maturation of microRNA-155 or -27a. In vitro RNA binding and Dicer inhibition assays suggested that the observed biological activity was most likely due to a sequence-specific PNA-RNA triplex formation that inhibited the activity of endonucleases responsible for microRNA maturation. The present study is the first example of modulation of activity of endogenous noncoding RNA using M-modified triplex-forming PNA.
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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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Effects of Modifying Thioflavin T at the N3-Position on Its G4 Binding and Fluorescence Emission. Reviewed International journal
Yuka Kataoka, Hiroto Fujita, Tamaki Endoh, Naoki Sugimoto, Masayasu Kuwahara
Molecules (Basel, Switzerland) 25 ( 21 ) 2020.10
We previously synthesized thioflavin T (ThT) with a hydroxyethyl group introduced at the N3-position (ThT-HE), which binds predominantly to the parallel G-quadruplex (G4) structure found in c-Myc and emits strong fluorescence. In this study, to investigate the effects of introduced substituents on G4 binding and fluorescence emission, a ThT derivative in which the hydroxyl group of ThT-HE was replaced with an amino group (ThT-AE) was synthesized for the first time. Furthermore, three other N3-modified ThT derivatives (ThT-OE2, ThT-SP, and ThT-OE11) having different substituent structures were synthesized by the N-acylation of the terminal amino group of ThT-AE, and their G4-binding and emission properties were investigated. The results showed that, although ThT-AE shows binding selectivity depending on the type of G4, its emission intensity is significantly decreased as compared to that of ThT-HE. However, ThT-OE11, which features an 11-unit oxyethylene chain attached to the terminal amino group of ThT-AE, regained about one-half of the emission intensity of ThT-HE while retaining selectivity for G4s. Accordingly, ThT-OE11 may be used as a key intermediate for synthesizing the conjugates of G4 binders and probes.
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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 48 ( 21 ) 12042 - 12054 2020.7
Joint Work
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/gkaa572
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Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions. Reviewed International journal
Saptarshi Ghosh, Shuntaro Takahashi, Tatsuya Ohyama, Tamaki Endoh, Hisae Tateishi-Karimata, Naoki Sugimoto
Proceedings of the National Academy of Sciences of the United States of America 117 ( 25 ) 14194 - 14201 2020.6
Joint Work
The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (Tm) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.
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Signaling Aptamer Optimization through Selection Using RNA-Capturing Microsphere Particles. Reviewed International journal
Tamaki Endoh, Naoki Sugimoto
Analytical chemistry 92 ( 11 ) 7955 - 7963 2020.6
Joint Work
Authorship：Lead author, Corresponding author
An RNA signaling aptamer is composed of two units: a sensing aptamer that binds the input target molecule and a working aptamer that binds the output target molecule to result in a detectable signal. A conformational change of the signaling aptamer that induces an allosteric interaction with the output target molecule in response to the input target molecule depends on a junction region, which connects the two aptamer units. Efficient and effective optimization of the junction region remains a technical challenge. In this study, we demonstrate a simple strategy for optimizing the junction region through functional RNA selection using RNA-capturing microsphere particles. From approximately 0.2 million sequence variants, a signaling aptamer that enabled intracellular detection of S-adenosyl methionine with a high signal-to-noise ratio, which is approximately 2-fold higher relative fluorescence increment compared to the previously reported signaling aptamer, was obtained after single round of selection. The technology demonstrated here can be used to select RNA sequences that carry out specific functions in response to particular stimuli.
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RNA-capturing microsphere particles (R-CAMPs) for optimization of functional aptamers Invited Reviewed
Endoh T., Ohyama T., and Sugimoto N.
Small 15 ( 26 ) e1808062 2019.6
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Conformational dynamics of the RNA G-quadruplex and its effect on translation efficiency Invited Reviewed
Endoh T. and Sugimoto N.
Molecules 24 ( 8 ) E1613 2019.4
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Validation of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in molecular crowding condition Reviewed
Ghosh S., Takahashi S., Endoh T., Tateishi-Karimata H., Hazra S., and Sugimoto N.
Ncleic Acids Res. 47 ( 7 ) 3284 - 3294 2019.4
Joint Work
DOI： 10.1093/nar/gkz071
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Co-transcriptional molecular assembly results in a kinetically controlled irreversible RNA conformational switch Reviewed
Endoh T. and Sugimoto N.
Anal. Chem. 90 ( 19 ) 11193 - 11197 2018.10
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Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations Reviewed International coauthorship
M. Trajkovski,* T, Endoh,* H. Tateishi-Karimata, T. Ohyama, S. Tanaka, J. Plavec and N. Sugimoto [* Equal contribution]
46 4301 - 4315 2018.5
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Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins Reviewed International coauthorship
E. Natan, T. Endoh, L. Haim-Vilmovsky, T. Flock, G. Chalancon, J. T. S. Hopper, B. Kintses, P. Horvath, L. Daruka, G. Fekete, C. Pál, B. Papp, E. Oszi, Z. Magyar, J. A. Marsh, A. H. Elcock, M. N. Babu, C. V. Robinson, N. Sugimoto, and S. A. Teichmann
25 279 - 288 2018.3
Joint Work
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Synthetic Chlorophyll-A Derivatives Stabilize DNA G-Quadruplex Structures
Nagano Yasunobu, Endoh Tamaki, Ogasawara Shin, Sugimoto Naoki, Tamiaki Hitoshi
BIOPHYSICAL JOURNAL 114 ( 3 ) 599A 2018.2
Joint Work
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Crowding Shifts the FMN Recognition Mechanism of Riboswitch Aptamer from Conformational Selection to Induced Fit Reviewed
A. B. Rode, T. Endoh, and N. Sugimoto
2018
Joint Work
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Conformational Dynamics of mRNA in Gene Expression as New Pharmaceutical Target Invited Reviewed
T. Endoh and N. Sugimoto
17 ( 9 ) 817 - 832 2017.9
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tRNA Shifts the G-quadruplex–Hairpin Conformational Equilibrium in RNA towards the Hairpin Conformer Reviewed
A. B. Rode, T. Endoh, and N. Sugimoto
Angew. Chem. Int. Ed. 55 14315 - 14319 2016.11
Joint Work
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Triplex-forming PNA modified with unnatural nucleobases: the role of protonation entropy in RNA binding Reviewed International coauthorship
T. Endoh, C. Annoni, D. Hnedzko, E. Rozners, and N. Sugimoto
Phys. Chem. Chem. Phys. 18 32002 - 32006 2016.11
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Correction: Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing. International coauthorship
Annoni C, Endoh T, Hnedzko D, Rozners E, Sugimoto N
Chemical communications (Cambridge, England) 52 ( 91 ) 13417 - 13418 2016.11
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Triplex-forming peptide nucleic acid modified with 2-aminopyridine as a new tool for detection of A-to-I editing Reviewed International coauthorship
Annoni C, Endoh T, Hnedzko D, Rozners E, Sugimoto N
52 7935 - 7938 2016.6
Joint Work
DOI： 10.1039/C6CC02164F
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Site-specific control of silica mineralization on DNA using a designed peptide Reviewed
Ozaki M, Nagai K, Nishiyama H, Tsuruoka T, Fujii S, Endoh T, Imai T, Tomizaki KY, Usui K
Chem. Commun. 52 ( 21 ) 4010 - 4013 2016.3
Joint Work
DOI： 10.1039/c5cc07870a
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Mechanical insights into ribosomal progression overcoming RNA G-quadruplex from periodical translation suppression in cells Reviewed
Endoh T, Sugimoto N
Sci. Rep. 6 22719 2016.3
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Real-time monitoring of G‑quadruplex formation during transcription Reviewed
Endoh T, Rode AB, Takahashi S, Kataoka Y, Kuwahara M, Sugimoto N
Anal. Chem. 88 ( 4 ) 1984 - 1989 2016.2
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Nucleobase-modified PNA suppresses translation by forming a triple helix with a hairpin structure in mRNA in vitro and in cells Reviewed International coauthorship
Endoh T, Hnedzko D, Rozners E, Sugimoto N
Angew. Chem. Int. Ed. 55 ( 3 ) 899 - 903 2016.1
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Rational design and tuning of functional RNA switch to control an allosteric intermolecular interaction Reviewed
Endoh T, Sugimoto N
Anal. Chem. 87 7628 - 7635 2015.8
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Key tertiary interactions in FMN riboswitch aptamers required for ligand binding Reviewed
Rode A B, Endoh T, Sugimoto N
Bull. Chem. Soc. Jpn. 88 946 - 948 2015.7
Joint Work
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Tuning riboswitch-mediated gene regulation by rational control of aptamer ligand binding properties Reviewed
Rode A B, Endoh T, Sugimoto N
Angew. Chem. Int. Ed. 54 905 - 909 2015.1
Joint Work
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Aptamer-based universal fluorometric sensors based on allosteric modulation of RNA–peptide Interactions Reviewed
Endoh T, Sugimoto N
ChemMedChem 9 2045 - 2048 2014.9
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Unusual −1 ribosomal frameshift caused by stable RNA G-quadruplex in open reading frame Reviewed
Endoh T, Sugimoto N
Anal. Chem., 85 11435 - 11439 2013.12
Joint Work
Authorship：Lead author
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Unusual-1 Ribosomal Frameshift Caused by Stable RNA G-Quadruplex in Open Reading Frame
Tamaki Endoh, Naoki Sugimoto
ANALYTICAL CHEMISTRY 85 ( 23 ) 11435 - 11439 2013.12
Joint Work
Authorship：Lead author Publisher：AMER CHEMICAL SOC
Tertiary structures formed by mRNAs impact the efficiency of the translation reaction. Ribosomal frameshift is a well-characterized recoding process that occurs during translation elongation. Pseudoknot and stem-loop structures may stimulate frameshifting by causing a translational halt at a slippery sequence. In this study, we evaluated the efficiency of an unusual 1 frameshift caused by a noncanonical RNA G-quadruplex structure in mammalian cells. The reporter gene construct consisting of a fluorescent protein and Luciferase enabled evaluation of apparent and absolute values of the 1 frameshift efficiency and revealed significant increase of the efficiency by G-quadrupex forming potential sequence. In addition, berberine, a small molecule that binds to and stabilizes G-quadruplex structures, further increased the frameshift efficiency. These results indicate that the stable G-quadruplex structure stimulates the unusual -1 frameshift and has a potential to regulate the frameshift with its ligand.
DOI： 10.1021/ac402497x
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Real-time monitoring of DNA hybridization kinetics at living cell surfaces Reviewed
Rode A B, Endoh T, Tateishi-Karimata H, Takahashi S, Sugimoto N
Chem. Commun. 49 8444 - 8446 2013.8
Joint Work
DOI： 10.1039/c3cc42990c
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Stability of RNA quadruplex in open reading frame determines proteolysis of human estrogen receptor α Reviewed
Endoh T, Kawasaki Y, Sugimoto N
Nucleic Acids Res. 41 6222 - 6231 2013.7
Joint Work
Authorship：Lead author
mRNAの翻訳領域中に形成されるRNA四重鎖構造が要因となるヒトエストロゲンレセプターアルファのタンパク質切断
要旨：翻訳反応は、塩基配列に保存された情報を、タンパク質という機能性分子に変換する重要な過程である。翻訳反応過程では、リボソームがmRNA上を5′側から3′側へ移動しながらアミノ酸を連結していく。この過程は、単調に進むのではなく、様々な要因により速度が遅くなったり、一時的に停滞したりする。近年では、翻訳伸長反応の速度変化が、翻訳後タンパク質の構造形成に影響を及ぼしていることが示唆されつつある。我々は最近、翻訳領域中に形成されるRNA四重鎖構造が、翻訳伸長反応の進行を妨げることをin vitroとin cellにて見出した。RNA四重鎖構造については、mRNAの非翻訳領域に存在するものを中心に研究が進められており、四重鎖構造による翻訳伸長反応の抑制が、細胞内でどのような生理的役割を果たしているのかは定かではない。
本研究では、翻訳領域中に四重鎖構造を形成しうる遺伝子の候補としてヒトエストロゲンレセプターアルファ（ERα）に着目し、四重鎖構造がERαの発現挙動に与える影響を解析した。ERαはマルチドメインから形成されるタンパク質であり、四重鎖構造を形成しうる配列は、DNA結合ドメインとリガンド結合ドメインをコードする領域の中間に存在する。そのため、四重鎖構造による翻訳伸長反応の抑制が、両ドメインの構造形成に影響する可能性が考えられる。まず、ERαの翻訳領域に存在するグアニンに富んだ配列を合成し、in vitroで構造を評価した。その結果、パラレル型の四重鎖構造を形成することが確認された。そこで、翻訳されるアミノ酸配列を同じにして、様々な熱安定性を有する変異配列を作製した。これらの変異配列を有するERαの遺伝子を細胞に導入し、その発現をウェスタンブロッティングで評価した。その結果、形成される四重鎖構造の安定性に応じて、完全長のERαの翻訳産物の他に、分子量の小さいERαが産出されていることが明らかとなった。N末端もしくはC末端に蛍光タンパク質を融合したERαを発現させて評価したところ、分子量の小さい翻訳産物は、完全長のERαが産出されてから分解を受けて生じていることが明らかとなった。アミノ酸配列は全く同じであるにも関わらず、四重鎖構造の熱安定性に応じて分解産物が生じていたことから、翻訳伸長反応の抑制を介してERαの構造と分解挙動に影響したと考えらえる。このような結果を踏まえ、mRNAは、単にタンパク質のアミノ酸配列を規定するだけではなく、タンパク質の構造形成から分解に至るまでに関与する高次なメッセージを有している可能性が考えられる。DOI： 10.1093/nar/gkt286
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Efficacy of base-modification on target binding of small molecule DNA aptamers Reviewed
Imaizumi Y., Kasahara Y., Kitadume S., Ozaki H., Endoh T., Kuwahara M. and N. Sugimoto
J. Am. Chem. Soc. 135 9412 - 9419 2013.6
Joint Work
低分子化合物に結合するDNAアプタマーにおける塩基修飾の有効性
要旨：DNAやRNAの一部を人工的な化学構造で改変した修飾核酸は、天然の核酸分子よりも優れた機能性を発揮することができる。特に、特定のタンパク質に強く結合するアプタマーは、核酸医薬などへの応用が見込まれる。これまでに、天然核酸ではアプタマーを取得することが困難なタンパク質に対し、塩基部位にアミノ酸側鎖を修飾することで高親和性を有する修飾核酸アプタマーを取得できることが示されている。一方で、タンパク質と比較して相互作用に寄与できる化学構造が少ない低分子化合物の場合、塩基部位への化学修飾が高親和性を有するアプタマーの取得に有効であるかどうかは実証されていない。
本研究では、天然核酸と塩基修飾核酸から同一の標的分子に対するアプタマーを取得し、塩基修飾による低分子化合物への結合親和性の向上を実証することを試みた。アプタマーを取得する標的分子として、抗がん剤であるカンプトテシン（CPT）誘導体を選択した。塩基修飾を有するDNA（修飾DNAライブラリー）は、チミジンの5 位にN6-アミノエチルアデニンを修飾したヌクレオチドを合成し、DNAポリメラーゼによる伸長反応中に取り込ませることで作製した。修飾DNA ライブラリーおよび天然型DNA ライブラリーを用いて、CPT誘導体に特異的に結合する核酸アプタマーの取得をそれぞれ行った。そして、得られた修飾DNA アプタマー（CMA）および天然型DNA アプタマー（CDA）のCPTに対する結合特性を比較検討した。表面プラズモン共鳴測定装置（SPR）を用いて結合親和性を評価した結果、CDAのCPTに対する解離定数が1.1 μMであったのに対し、CMAは0.039 μMと30倍近くCPTに対する結合親和性が高くなっていた。また、CMAが有するCPTに対する結合親和性は、CPT以外の低分子化合物に対する天然型DNA/RNAアプタマーと比較しても高く、アプタマーに対する塩基修飾の有効性を示すことができた。さらに、塩基修飾による結合親和性の増大は解離速度定数が0.00033 s-1という非常に小さい値を示すことが原因であり、CDAと比較して270倍遅い解離速度を示すことが明らかとなった。このことから、本研究で取得されたCPTに対する修飾塩基アプタマーを利用し、がん細胞への効率的なドラッグデリバリーなどへの応用が見込まれる。DOI： 10.1021/ja4012222
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Translational halt during elongation caused by G-quadruplex formed by mRNA Invited Reviewed
Endoh T, Kawasaki Y, Sugimoto N
Methods 64 73 - 78 2013.6
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Suppression of gene expression by G-quadruplexes in open reading frames depends on g-quadruplex stability Reviewed
Endoh T, Kawasaki Y, Sugimoto N
Angew. Chem. Int. Ed. 52 5522 - 5526 2013.5
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Selection of RNAs for constructing “Lighting-UP” biomolecular switches in response to specific small molecules Reviewed
Endoh T, Sugimoto N
PLoS ONE 8 e60222 2013.3
Joint Work
Authorship：Lead author
生体内では、様々な分子が、分子間のアロステリックな相互作用を介して機能発現を調節している。例えば、リボスイッチと呼ばれる機能性RNAは、特定の代謝産物に応答して遺伝子の発現を調節する。特に、翻訳反応の調節に関わるリボスイッチの場合、mRNA上に存在するリボスイッチが構造変化することで、リボソームとmRNAとの相互作用がアロステリックに制御されている。このようなメカニズムは、RNAと特定分子との結合に伴うRNAの構造変化が、タンパク質機能の調節に有用であることを示している。
本研究では、特定の低分子化合物を検出するために、RNA‒タンパク質間のアロステリック相互作用を利用した発光バイオセンサーを構築した。我々はこれまでに、RNAの検出を目的とした遺伝子改変型のルシフェラーゼを構築しており、特定のRNAとの結合を発光シグナルで検出することに成功している。そこで、低分子化合物との結合によるRNA構造変化を起因とし、改変型ルシフェラーゼと結合できるようになるRNAを構築することにした。喘息薬の一種であるテオフィリン、および抗生物質の一種であるテトラサイクリンを検出対象とし、これらの分子に特異的に結合することが知られるRNAアプタマーを利用した。それぞれのRNAアプタマーと、改変型ルシフェラーゼとの結合に必要なTAR-RNAとをランダムな配列を介して連結したRNAライブラリーを設計した。そして、テオフィリン、テトラサイクリンに応答して、TAR-RNAの結合標的であるTatペプチドと相互作用するRNAをセレクションにより獲得した。6サイクルのセレクション過程を経たのちに得られたRNAは、それぞれの標的分子に応答してTatペプチドとの結合定数が10倍以上上昇した。そこで、生体外翻訳系を用いて発現させた改変型ルシフェラーゼと、セレクションにより得られたRNAとを混合し、発光シグナルによる標的分子の検出を試みた。その結果、標的分子の濃度に依存した発光シグナルの上昇が確認でき、テオフィリン10 μM、テトラサイクリン2 μMでも有意に検出可能であった。このことは、RNAによる分子認識と、それに引き続くタンパク質とのアロステリックな相互作用を介して、ルシフェラーゼの発光反応触媒機能を調節できたことを示している。RNAとタンパク質はどちらも細胞内で発現させることが可能であり、細胞内で機能する発光バイオセンサーへの応用が期待される。 -
Dehydration from conserved stem regions is fundamental for ligand-dependent conformational transition of the adenine-specific riboswitch Invited Reviewed
Kumar V, Endoh T, Murakami K, Sugimoto N
Chem. Commun. 48 9693 - 9695 2012.9
Joint Work
DOI： 10.1039/C2CC34506D
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Synchronized translation for detection of temporal stalling of ribosome during single-turnover translation Reviewed
Endoh T, Kawasaki Y, Sugimoto N
Anal. Chem. 84 857 - 861 2012.1
Joint Work
Authorship：Lead author
翻訳伸長反応は一律に進むのではなく、様々な因子の影響を受け、その速度が大きく変化する。特に、mRNA中に存在するレアコドンや安定な二次構造は、翻訳伸長反応速度を低減させることが知られている。近年、翻訳伸長反応の一時的な停滞や速度変化が、タンパク質の構造形成過程に大きな影響をもたらすことが明らかになりつつある。つまりmRNAは、ただ単にアミノ酸配列を規定するだけではなく、翻訳伸長反応速度への影響を介して、タンパク質の機能発現までも調節している可能性がある。そのため、mRNA上のどの位置で、どの程度、翻訳伸長反応が停滞したり速度変化を示したりするのかを正確に解析することが重要である。しかしながら、翻訳反応過程では、律速となる反応が翻訳開始過程にあることが知られており、翻訳伸長反応のみに焦点を当てた解析は、既存の解析手法では困難である。
本研究では、翻訳伸長反応のみに焦点を当て、反応過程の継時的な解析を可能にするSynchronized Translation（同調翻訳）という解析手法を構築した。一段階目の反応として、再構成型の無細胞翻訳反応系を利用し、翻訳反応溶液から特定のアミノアシルtRNA合成酵素を取り除いておくことで、翻訳を開始したリボソームを特定のコドンの位置で強制的に停止させた。また、この反応段階において非天然蛍光アミノ酸を翻訳産物に導入し、翻訳産物を蛍光シグナルで解析できるようにした。二段階目の反応として、翻訳反応に必要な全ての因子を含む反応溶液を加え、翻訳伸長反応を一段階目で停止させてある位置から再開させた。これにより、律速段階となる翻訳開始過程を省き、1分間というこれまでにはない短い時間軸における経時的な翻訳伸長反応の解析を可能にした。結果として、46アミノ酸を伸長する間に3カ所で翻訳伸長反応が一時的に停滞し、そのうちの2カ所はレアコドンが原因となっていることが明らかとなった。DOI： 10.1021/ac202712g
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Photosensitizing carrier proteins for photoinducible RNA interference Reviewed
Matsushita-Ishiodori Y, Kuwabara R, Sakakoshi H, Endoh T, Ohtsuki T
Bioconjug. Chem. 22 2222 - 2226 2011.10
Joint Work
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Direct detection of RNAs in living cells using peptide-inserted Renilla luciferase Reviewed
Andou T, Endoh T, Mie M, Kobatake E.
Analyst 136 2446 - 2449 2011.5
Joint Work
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Gene regulation system with an artificial RNA switch operating in human cells Reviewed
Endoh T, Sugimoto N.
Chembiochem. 2011 Apr 29. [Epub ahead of print] 2011.4
Joint Work
Authorship：Lead author
近年、リボスイッチと呼ばれる機能性RNAを利用した特徴的な遺伝子発現制御機構の存在が明らかになってきている。リボスイッチは主に、mRNAの5’非翻訳領域中に存在しており、標的となる代謝産物の結合に伴うRNAの構造変化を介して遺伝子発現を調節している。これまで、原核生物では多種類のリボスイッチが確認されているものの、真核生物ではごく少数の生物種に限られており、特にヒトを中心とした高等生物では報告例がない。一方で、リボスイッチに類似した機構による人工的な遺伝子発現制御システム（人工リボスイッチ）がいくつか開発されているものの、これまでのところ転写後反応の制御に機能する人工リボスイッチに関する報告のみであった。
本研究では、特異的なRNA配列（TAR-RNA）に結合して遺伝子の転写を活性化する免疫不全ウイルス由来のトランス活性化因子（Tat）を利用し、ヒト細胞内での遺伝子発現を転写段階で制御できる人工リボスイッチシステムの構築を行った。TAR-RNAのループ部位に対して、低分子化合物（テオフィリン）に結合するRNAアプタマーを挿入した人工RNAを設計し、これをもとにレポーターベクターを構築した。そして、作製したベクターを、Tatを発現するベクターと共にHeLa細胞に導入し、テオフィリン存在下での遺伝子発現を評価した。その結果、0 – 1 mMの範囲において、培地中に添加したテオフィリン濃度に依存して遺伝子発現が抑制されることが示された。細胞外の実験結果から、テオフィリン存在下において設計したRNAとTatとの結合定数が減少することが示されているため、TAR-RNAに挿入したアプタマーとテオフィリンとの結合がTatの結合および転写の活性化を阻害していると考えられる。 -
Development of an RNA detection system using bioluminescence resonance energy transfer Reviewed
Andou T, Endoh T, Mie M, Kobatake E
Sensor Actuat. B Chem. 152 ( 2 ) 277 - 284 2011.3
Joint Work
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Evaluation of small ligand-protein interactions by using T7 RNA polymerase with DNA-modified ligand Reviewed
Mie M, Sugita R, Endoh T, Kobatake E.
Anal. Biochem. 405 ( 1 ) 109 - 113 2010.6
Joint Work
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Cellular siRNA delivery using TatU1A and photo-induced RNA interference Invited
Endoh T, Ohtsuki T
Methods Mol. Biol. 623 271 - 281 2010.4
Joint Work
Authorship：Lead author
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Improvement of CPP-RBD for siRNA Delivery
Kuwabara Rina, Endoh Tamaki, Sisido Masahiko, Ohtsuki Takashi
ANIMAL CELL TECHNOLOGY: BASICS & APPLIED ASPECTS 16 191 - 196 2010
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Detection of bioactive small molecules by fluorescent resonance energy transfer (FRET) in RNA-protein conjugates Reviewed
Endoh T, Shintani R, Mie M, Kobatake E, Ohtsuki T, Sisido M.
Bioconjug. Chem. 20 2242 - 2246 2009.12
Joint Work
Authorship：Lead author
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PNA Arrays for miRNA Detection Reviewed
Endoh T, Kitamatsu M, Sisido M, Ohtsuki T
Chem. Let. 38 438 - 439 2009.5
Joint Work
Authorship：Lead author
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Carrier PNA for shRNA delivery into cells Reviewed
Kitamatsu M, Kubo T, Matsuzaki R, Endoh T, Ohtsuki T, Sisido M
Bioorg. Med. Chem. Lett. 19 3410 - 3413 2009.5
Joint Work
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Spatial regulation of specific gene expression through photoactivation of RNAi Reviewed
Endoh T, Sisido M, Ohtsuki T
J. Control. Release 137 241 - 245 2009.4
Joint Work
Authorship：Lead author
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APPLICATIONS OF PEPTIDE NUCLEIC ACIDS FOR RNA PURIFICATION, RNA DETECTION, AND INTRACELLULAR RNA DELIVERY
Takashi Ohtsuki, Tamaki Endoh, Mizuki Kitamatsu, Masahiko Sisido
IFPT'6: PROGRESS ON POST-GENOME TECHNOLOGIES, PROCEEDINGS 22 - 23 2009
Joint Work
Publisher：SOUTHEAST UNIV PRESS
Peptide nucleic acids (PNAs) are nucleic acid analogs with an achiral polyamide backbone. Since the PNA-RNA hybrids are thermally more stable than the corresponding DNA-RNA or RNA-RNA hybrids, PNAs can be tools for catching and detecting RNAs. In this study, we developed PNA-based methods for RNA isolation, RNA detection, and intracellular RNA delivery.
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Direct detection of RNA transcription by FRET imaging using fluorescent protein probe Reviewed
Endoh T, Mie M, Kobatake E
J. Biotechnol., 133 413 - 417 2008.11
Joint Work
Authorship：Lead author
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RNA detection using peptide-inserted Renilla luciferase Reviewed
Andou T, Endoh T, Mie M, Kobatake E
Anal. Bioanal. Chem. 393 661 - 668 2008.11
Joint Work
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Cellular siRNA delivery mediated by a cell-permeant RNA-Binding protein and photoinduced RNA interference Reviewed
Endoh Tamaki, Sisido Masahiko, Ohtsuki Takashi
BIOCONJUGATE CHEMISTRY 19 ( 5 ) 1017 - 1024 2008.5
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Cellular siRNA delivery mediated by cell-penetrating RNA-binding protein and photo-induced RNA interference Reviewed
Endoh T, Sisido M, Ohtsuki T
Bioconjug. Chem., 19 1017 - 1024 2008.4
Joint Work
Authorship：Lead author
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Photo inducible RNA interference using cell permeable protein carrier
Endoh T, Sisido M, Ohtsuki T
Nucleic Acids Symp. Ser., 51 127 - 128 2007.11
Joint Work
Authorship：Lead author
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Construction of intramolecular luciferase complementation probe for detecting specific RNA Reviewed
Endoh Tamaki, Mie Masayasu, Funabashi Hisakage, Sawasaki Tatsuya, Endo Yaeta, Kobatake Eiry
BIOCONJUGATE CHEMISTRY 18 ( 3 ) 956 - 962 2007.5
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Construction of intramolecular luciferase complementation probe for detecting specific RNA Reviewed
Endoh T, Mie M, Funabashi H, Sawasaki T, Endo Y, Kobatake E
Bioconjug. Chem., 18 956 - 962 2007.3
Joint Work
Authorship：Lead author
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Tamaki Endoh, Hisakage Funabashi, Masayasu Mie, Eiry Kobatake
Analytical Chemistry 77 ( 14 ) 4308 - 4314 2005.7
Joint Work
Detection of specific nucleic acids is important to understand cellular mechanisms and functions of gene regulation. Here, we demonstrated a novel method to detect specific nucleic acids using recombinant protein and oligonucleotides. A recombinant protein YRGnC-11ad, which has a Rev-peptide between enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) was constructed and expressed in HeLa cells. Rev-peptide, which corresponds to amino acids 34-50 of the HIV-1 Rev protein, indicates disordered structure in solution but forms α-helical and elongated conformation upon binding to Rev response element RNA (RRE-RNA) and Rev-aptamer, respectively. We confirmed that YRGnC-11ad could specifically bind to RRE-RNA and Rev-aptamer in cell lysate, and fluorescent resonance energy transfer (FRET) signal was changed upon binding following the conformational change of Rev-peptide. To utilize this FRET signal change toward the detection of specific nucleic acids, we split the RRE-RNA sequence and connected to the complementary oligonucleotide for target nucleic acids. When each two oligonucleotides hybridized to an adjacent region of target nucleic acids correctly, a Rev-peptide binding site was reformed on the hybridized complex. And we could confirm that YRGnC-11ad recombinant protein indicated FRET increase upon binding to the hybridized complex in cell lysate. These results suggest that the recombinant protein probe is available for specific nucleic acid detection. © 2005 American Chemical Society.
DOI： 10.1021/ac048491j
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Novel method for detection of specific nucleic acids by recombinant protein with FRET Reviewed
Endoh T, Funabashi H, Mie M, Kobatake E
Anal. Chem., 77 4308 - 4314 2005.7
Joint Work
Authorship：Lead author
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Induction of neural differentiation by electrically stimulated gene expression of NeuroD2. Reviewed
Mie M, Endoh T, Yanagida Y, Kobatake E, Aizawa M
J. Biotechnol., 100 231 - 238 2003.2
Joint Work