Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We demonstrate that liposomes with diameters of more than 10 μm (giant unilamellar vesicles, GUVs) self-aggregate in a non-crosslinking manner by forming DNA duplexes with blunt ends on their surfaces much more sensitively than gold nanoparticles (AuNPs) by virtue of their unique features.

DOI： 10.1093/chemle/upaf012

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Artificial riboswitches responsive to user-defined analytes can be constructed by successfully inserting in vitro selected aptamers, which bind to the analytes, into untranslated regions of mRNA. Among them, eukaryotic riboswitches are more promising as biosensors than bacterial ones because they function well at ambient temperature. In addition, cell-free expression systems allow the broader use of these riboswitches as cell-free biosensors in an environmentally friendly manner without cellular limitations. The current best cell-free eukaryotic riboswitch regulates eukaryotic canonical translation initiation through self-cleavage mediated by an implanted analyte-responsive ribozyme (i.e., an aptazyme, an aptamer-ribozyme fusion). However, it has critical flaws as a sensor: due to the less-active ribozyme used, self-cleavage and translation reactions must be conducted separately and sequentially, and a different aptazyme has to be selected to change the analyte specificity, even if an aptamer for the next analyte is available. We here stepwise engineered novel types of cell-free eukaryotic riboswitches that harness highly active self-cleavage and thus require no reaction partitioning. Despite the single-step and one-pot reaction, these riboswitches showed higher analyte dose dependency and sensitivities than the current best cell-free eukaryotic riboswitch requiring multistep reactions. In addition, the analyte specificity can be changed in an extremely facile way, simply by aptamer substitution (and the subsequent simple fine-tuning for giant aptamers). Given that cell-free systems can be lyophilized for storage and transport, the present one-pot and thus easy-to-handle cell-free biosensors utilizing eukaryotic riboswitches are expected to be widely used for on-the-spot sensing of analytes at ambient temperature.

DOI： 10.1021/acssynbio.4c00341

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Cold Spring Harbor Laboratory  

In general, riboswitches functioning through a cotranscriptional kinetic trapping mechanism (kt-riboswitches) show higher switching efficiencies in response to practical concentrations of their ligand molecules than eq-riboswitches, which function by an equilibrium mechanism. However, the former have been much more difficult to design due to their more complex mechanism. We here successfully developed a rational strategy for constructing eukaryotic kt-riboswitches that ligand-dependently enhance translation initiation mediated by an internal ribosome entry site (IRES). This was achieved both by utilizing some predicted structural features of a highly efficient bacterial kt-riboswitch identified through screening and by completely decoupling an aptamer domain from the IRES. Three kt-riboswitches optimized through this strategy, each responding to a different ligand, exhibited three- to sevenfold higher induction ratios (up to ∼90) than previously optimized eq-riboswitches regulating the same IRES-mediated translation in wheat germ extract. Because the IRES used functions well in various eukaryotic expression systems, these types of kt-riboswitches are expected to serve as major eukaryotic gene regulators based on RNA. In addition, the present strategy could be applied to the rational construction of other types of kt-riboswitches, including those functioning in bacterial expression systems.

DOI： 10.1261/rna.079778.123

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Language：English   Publishing type：Research paper (scientific journal)  

Gold nanoparticles (AuNPs) have been utilized as colorimetric biosensors, where target molecule-induced AuNP aggregation can be recognized by a colour change from red to blue. Particularly, single-stranded DNA (ssDNA)-immobilized AuNPs (ssDNA-AuNPs) have been applied to genetic diagnosis due to their rapid and sequence-specific aggregation properties. However, the effect of the density of immobilized ssDNA have not been investigated yet. In this study, we developed a method to control the amount of immobilized ssDNA by use of ethylene glycol, which is expected to control the ice crystal spacing in a freezing-thawing ssDNA-AuNP synthesis method. We also investigated the effect of the DNA density on the sensitivity of the target ssDNA detection, and found that the detection sensitivity was improved at lower DNA densities. To discuss the reason for the improved detection sensitivity, we modified the ssDNA-AuNPs with alkane thiol for better dispersion stability against salt. The results suggest that the DNA density, rather than the dispersion stability, has a significant impact on detection sensitivity.

DOI： 10.1039/D3RA06528F

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and cis-acting gene regulatory RNA. Although such an aptamer can be obtained through in vitro selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson–Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.

DOI： 10.1021/acssynbio.2c00475

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We chose two types of mid-sized Arg-rich peptides (Rev-pep and Tat-pep) as ligands and used their aptamers to construct efficient eukaryotic ON-riboswitches (ligand-dependently upregulating riboswitches). Due to the aptamers’ high affinities, the best Rev-pep-responsive and Tat-pep-responsive riboswitches obtained showed much higher switching efficiencies at low ligand concentrations than small ligand-responsive ON-riboswitches in the same mechanism. In addition, despite the high sequence similarity of Rev-pep and Tat-pep, the two best riboswitches were almost insensitive to each other’s peptide ligand. Considering the high responsiveness and specificity along with the versatility of the expression platform used and the applicability of Arg-rich peptides, this orthogonal pair of riboswitches would be widely useful eukaryotic gene regulators or biosensors.

DOI： 10.1016/j.bmcl.2022.128839

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We successfully constructed a coupled in vitro transcription/translation (cIVTT) system based on wheat germ extract (WGE) for efficient expression from PCR-generated DNA templates in short-time (∼3-h) batch reactions. The productivity of this system under optimized conditions was 85 μg (2.8 nmol) per 1 mL of reaction solution (corresponding to 425 μg per 1 mL of WGE), which was about 9-fold higher than that by the conventional batch method using mRNA as a template. The DNA template concentration required for efficient cIVTT was as low as 2.5 nM, which is much lower than those required for other eukaryotic cIVTT systems to maximize their productivity (30–50 nM). The productivity of the present system with a 2.5 nM template was 80-fold and 4-fold higher than that of a commercially available WGE-based cIVTT system with a 2.5 nM and a 40 nM template, respectively. In addition, the present system functioned well in a liposome (i.e., in an artificial cell) without a loss of productivity. Given that WGE-based systems have the advantage of being suitable for the expression of a broad range of proteins, the present cIVTT system is expected to be widely used in future cell-free synthetic biology.

DOI： 10.1016/j.bmcl.2021.128412

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Bio-Protocol, LLC  

Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes, the spontaneous emulsion transfer method is superior to others in that it allows us to readily prepare relatively large liposomes whose sizes are controlled (from micrometer- to millimeter-sized liposomes) without special equipment. However, conventional protocols for this method require liposomes to contain a considerably high concentration of sucrose (high-density solute), which severely inhibits gene expression, one of the most important biochemical reactions. Thus, we optimized the preparation conditions to develop a wheat germ extract (WGE)-based protocol that requires a much lower concentration of sucrose and has almost no effect on eukaryotic cell-free translation. Our protocol allows us to successfully prepare millimeter-sized, moderately stable, WGE-encapsulating liposomes in which WGE translation takes place efficiently. Since a broad range of genes derived from various types of organisms can be efficiently translated in a WGE-based translation system, liposomes prepared using our protocol would be useful as a versatile research tool for artificial cells.

DOI： 10.21769/bioprotoc.4054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Gold nanoparticles (AuNPs) are often used for biosensing. In particular, aptamer-modified AuNPs are often used for colorimetric molecular detection, where target molecule-induced AuNP aggregates can be recognized by a color change from red to blue. However, non-specific aggregation could be induced by various compounds, leading to false-positive results. In this work we employed high-density ssDNA modification on the AuNP surface to prevent non-specific aggregation. The covalently immobilized DNA brush was used as an anchor for an aptamer specific for the target molecule. Herein, as a proof-of-concept study, we demonstrated detection of estradiol (E2), one of the endocrine-disrupting estrogen molecules as a model target, in the presence of antibiotic kanamycin (KN) as a model of co-contaminating compounds that induce non-specific aggregation of AuNPs. We also developed a smartphone dark field microscope (DFM) to visualize AuNP aggregation. Our previous study demonstrated that the observation of light scattering by AuNP aggregates with DFM can be applied for versatile molecular detection. In this work, we could successfully detect E2 with the smartphone DFM, and the results were verified by the results from a conventional benchtop DFM. This study would contribute to the future field applicability of AuNP-based sensors.

DOI： 10.1039/d0ra05149g

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Authorship：Lead author,　Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Analytical Chemistry  

A nonsense suppressor tRNA (sup-tRNA) allows a natural or non-natural amino acid to be assigned to a nonsense codon in mRNA. Sup-tRNAs were utilized initially for studying tRNA functions but lately are used more for protein engineering and gene regulation. In the latter application, a sup-tRNA that is aminoacylated with a natural amino acid by the corresponding aminoacyl-tRNA synthetase is used to express a full-length natural protein from its mutated gene with a nonsense codon in the middle. This type of sup-tRNA has recently been artificially evolved to develop biosensors. In these biosensors, an analyte induces the processing of an engineered premature sup-tRNA into a mature sup-tRNA, which suppresses the corresponding nonsense codon incorporated into a gene, encoding an easily detectable reporter protein. This review introduces sup-tRNA-based biosensors that the author’s group has developed by utilizing bacterial and eukaryotic cell-free translation systems.

DOI： 10.2116/analsci.20scr01

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

We here designed an in vitro selection scheme for obtaining an aptamer with which to rationally construct an artificial riboswitch as its component part. In fact, a nanosized DNA-binding aptamer obtained through this scheme allowed us to easily and successfully create eukaryotic riboswitches that upregulate internal ribosome entry site-mediated translation in response to the ligand (nanosized DNA) in wheat germ extract, a eukaryotic cell-free expression system. The induction ratio of the best riboswitch ligand-dose-dependently increased to 21 at 300 μM ligand. This switching efficiency is much higher than that of the same type of riboswitch with a widely used theophylline-binding aptamer, which was in vitro selected without considering its utility for constructing riboswitches. The selection scheme described here would facilitate obtaining various ligand/aptamer pairs suitable for constructing artificial riboswitches, which could serve as elements of synthetic gene circuits in synthetic biology.

DOI： 10.1021/acssynbio.0c00384

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

C promoter binding factor 1 (CBF1) (alias RBPJ) is a critical transcription factor involved in Notch signaling. The activation of Notch signaling through CBF1 maintains the angiostatic state of endothelial cells suppressing angiogenesis, that is, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) induces angiogenesis by promoting the proteasomal degradation of CBF1, in addition to endothelial cell proliferation. To date, angiogenic inhibitors targeting VEGF have been successfully used in clinics for cancer and age-related macular degeneration. Most antiangiogenic drugs, however, only target VEGF or VEGF receptors. In this study, to expand the repertoire of antiangiogenic therapeutics, we developed 15 single-stranded deoxyribonucleic acid (ssDNA) aptamers capable of binding to CBF1 with high affinity (Kd; 10-300 nM). To this end, systematic evolution of ligands by the exponential enrichment (SELEX) method was applied. One of the CBF1-binding ssDNA aptamers, Apt-3, inhibited angiogenesis through the activation of Notch signaling in vitro. We found that Apt-3 directly interacted with the LAG1 domain of CBF1. We suggest that the Apt-3 ssDNA aptamer may contribute to the development of a novel angiogenic inhibitor, which does not target VEGF.

DOI： 10.1089/nat.2020.0875

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

We sought to prepare millimeter-sized supergiant unilamellar vesicles (SGUVs) by spontaneous emulsion transfer for efficient, eukaryotic cell-free translation in the interior. Although the conventional protocols require that a considerably high concentration of sucrose be encapsulated into the SGUVs for their efficient formation, such high amounts of sucrose severely inhibited cell-free translation based on wheat germ extract (WGE). We thus optimized the preparation conditions to permit SGUV formation at a much lower concentration of sucrose that has almost no effect on WGE translation. Under the optimized conditions, we successfully prepared WGE translation system-encapsulating SGUVs that allow for protein synthesis with a high efficiency comparable to that outside a liposome. The optimization also resulted in a high rate of successful SGUV formation (>90%) and a decent stability of the formed SGUVs (>60 min). These SGUVs are expected to serve as research tools in cell-free synthetic biology and as foundations for artificial cell-based biosensors.

DOI： 10.1021/acssynbio.0c00173

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Wheat germ extract (WGE) is one of the most widely used eukaryotic cell-free translation systems for easy synthesis of a broad range of proteins merely by adding template mRNAs. Its productivity has thus far been improved by removing translational inhibitors from the extract and stabilizing the template with terminal protectors. Nonetheless, there remains room for increasing the yield by designing a terminally protected template with higher susceptibility to translation. Given the fact that a 5′ terminal protector is a strong inhibitor of the canonical translation, we herein focused on Cripavirus internal ribosome entry sites (IRESes), which allow for a unique translation initiation from a non-AUG start codon without the help of any initiation factors. We mutated their start codons to enhance the IRES-mediated translation efficiency in WGE. One of the mutants showed considerably higher efficiency, 3–4-fold higher than that of its wild type, and also 3–4-fold higher than the canonical translation efficiency by an IRES-free mRNA having one of the most effective canonical-translation enhancers. Because this mutated IRES is compatible with different types of genes and terminal protectors, we expect it will be widely used to synthesize proteins in WGE.

DOI： 10.1016/j.bmcl.2019.126729

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Wheat cell-free expression systems based on wheat germ extract (WGE) enable us to briefly synthesize various types of proteins in vitro merely by exogenously adding their mRNA templates. Moreover, it is possible to produce larger amounts of protein by thoroughly removing the endosperm, which contains many translation inhibitors, including ribonucleases (RNases). However, because small amounts of RNases are also present even in an endosperm-free, high-quality WGE (hqWGE), the in-vitro transcribed mRNA is rapidly degraded. In particular, 3′ exonucleases have been considered as the major RNases that degrade mRNA. We thus herein performed in vitro selection to find an effective, short 3′ protector sequence from a random RNA pool. The selected sequences stabilized in vitro transcripts in the hqWGE more effectively than the previously reported, longer 3′ protectors did. In addition, when one of these 3′ protectors was minimized and then fused to mRNA, the translation efficiency increased 5–6-fold in the hqWGE, mainly due to the mRNA stabilization.

DOI： 10.1016/j.bmcl.2019.06.058

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Language：English   Publishing type：Research paper (scientific journal)  

Gold nanoparticles (AuNPs) have been commonly used in molecular sensing, in the form of observation of the color change from red to blue of the AuNP solution, caused by target-molecule-induced AuNP aggregation. In this work, the changes in absorbance and scattering spectra caused by AuNP aggregation were studied using thrombin-induced AuNP aggregation as a model. We demonstrated for the first time that scattering spectra is more sensitive to the changes owing to AuNP aggregation than absorbance spectra. Moreover, a digital color analysis of darkfield images using dark field microscopy (DFM) facilitated a simple method for detection of AuNPs aggregation without the use of spectroscopic analysis. Furthermore, we demonstrated that DFM is useful for detecting AuNPs aggregation in a colored solution, in which the color change by AuNPs aggregation is not visible.

DOI： 10.2116/analsci.18P571

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Biotechnology  

Many cyanophages, which infect cyanobacteria, most of possess putative sigma factors that have high amino acid sequence homology with the σ70-type sigma factor present in cyanobacteria, allowing them to obtain energy and metabolites for their own propagation. In this study, we aimed to modify the carbon metabolism of Synechococcus elongatus PCC 7942 by expressing putative sigma factors from Synechococcus phages to improve bioproduction. Four cyanophage-derived putative sigma factors—putative RpsD4 from Synechococcus phage S-CBS1, putative RpoD and putative RpoS from S-CBS2, and putative RpsD4 from S-CBS3—were selected for this purpose. These were introduced into S. elongatus PCC 7942, and their expression was controlled with a theophylline-dependent riboswitch. The expression of the putative RpoD from S-CBS2 and putative RpsD4 from S-CBS3 resulted in a significant decrease in the growth rate of S. elongatus PCC 7942. In addition, metabolome analysis showed a 3.2-fold increase in acetyl-CoA concentration with the expression of the putative RpoD from S-CBS2 and a 1.9-fold increase with the putative RpsD4 from S-CBS3. The results of RT-qPCR showed that several sugar metabolism genes were repressed by the putative RpoD and activated by the putative RpsD4. In particular, the engineered strain overexpressing the putative RpsD4 and expressing phosphate acetyltransferase succeeded in improving the productivity of the model target product acetate to 217% of its previous value. To the best of our knowledge, this study is the first to modify the metabolism of S. elongatus PCC 7942 by expressing their putative sigma factors from cyanophages.

DOI： 10.1016/j.jbiosc.2018.07.019

CiNii Books

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Ltd  

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5′ untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5′ end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5′ end, and this suppression is dependent on the structure's stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5′-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.

DOI： 10.1016/j.bmcl.2018.06.041

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5' cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.

DOI： 10.1021/acssynbio.7b00124

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have rationally constructed a novel regulation-type of artificial riboswitch that ligand-dose dependently upregulates translation initiation mediated by a 30 cap-independent translation element (30 CITE) with no major hybridization switches in a plant expression system (wheat germ extract).

DOI： 10.1039/c6mb00748a

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2016.05.091

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 mu g mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.

DOI： 10.1039/c5ob02533h

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：ELSEVIER ACADEMIC PRESS INC  

A number of natural and artificial bacterial riboswitches have been reported thus far. However, they generally function only in bacteria, not in eukaryotes. This is because of the differences of expression mechanisms (transcription, translation, and so on) between these two main types of organisms. For example, the mechanism of translation initiation is quite different between bacteria and eukaryotes, especially in ribosome loading on mRNA. While the bacterial ribosome binds to a well-conserved, internal sequence some bases before the start codon to initiate translation, the eukaryotic one is loaded on the 50 terminus with the help of certain eukaryotic initiation factors. This means not only that bacterial riboswitches regulating translation initiation are not available in eukaryotic translation systems, but also that it is physically difficult to construct eukaryotic ON riboswitches that regulate the eukaryotic canonical translation initiation, because an aptamer cannot be inserted upstream of the ribosome loading site. However, the mechanism of noncanonical translation initiation via "ribosomal shunt" enables us to design translation initiation-modulating (specifically, ribosomal shunt-modulating) eukaryotic ON riboswitches. This chapter describes a facile method for engineering these ribosomal shunt-modulating eukaryotic ON riboswitches by using a cell-free translation system. Because these riboswitches do not require hybridization switching thanks to a unique shunting mechanism, they have the major advantages of a low energy requirement for upregulation and relatively straightforward design over common hybridization switch-based ON riboswitches.

DOI： 10.1016/bs.mie.2014.10.033

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.

DOI： 10.1039/c5ob00794a

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We investigated the end processing and degradation of premature tRNAs in wheat germ extract (WGE), which led to the discovery of end protectors useful for stabilizing an in vitro transcript against various ribonucleases and thereby enhancing its apparent activity in WGE.

DOI： 10.1039/c4ob02221a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 106 M. The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.

DOI： 10.1021/ac503522f

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

High-quality wheat germ extract (hqWGE) is very useful for the high-yield production of various types of protein. The most important key to high productivity is the design of mRNA templates. Although the design has been refined for straightforward and efficient translation in hqWGE, there is still room for improvement in untranslated regions (UTRs), especially the 3' UTR length, because a long, cumbersome 3' UTR is commonly used for translation enhancement. Here we examined some short viral 3' cap-independent translation enhancers (3' CITEs) to identify effective ones for efficient translation in hqWGE. We then combined the most effective 3' CITE and a 5' enhancer to further increase the translation efficiency. mRNA with the optimal short 3' and 5' UTRs, both of whose length was less than 150 nt, exhibited a productivity of 1.4 mg/mL in prolonged large-scale protein synthesis in hqWGE, which was comparable to that of control mRNA with a commonly-used long 30 UTR (similar to 1200 nt). (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2014.07.004

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

Riboswitches are composed of two regions: one for binding to the ligand (the aptamer domain) and the other for regulating the expression of the gene (the expression platform). In most riboswitches (both natural and artificial), a part of the aptamer domain required for ligand binding is directly involved in the regulation of expression, so that it is difficult to design other ligand-responsive riboswitches based on these riboswitches even by using artificial aptamers obtained through in vitro selection. This chapter describes a method for rationally constructing a foundational ON-riboswitch, which is easily available for the design of other ligand-dependent riboswitches, by introducing a new region (a modulator sequence: MS) in addition to the two basic regions. A facile method for preparing arbitrary molecule-dependent riboswitches based on the foundational riboswitch is also presented. © Springer Science+Business Media New York 2014.

DOI： 10.1007/978-1-62703-755-6_12

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Language：English   Publishing type：Research paper (scientific journal)  

The cyanobacterium Synechococcus elongatus PCC 7942 is a major model species for studies of photosynthesis. It is are also a potential cell factory for the production of renewable biofuels and valuable chemicals. We employed engineered riboswitches to control translational initiation of target genes in this cyanobacterium. A firefly luciferase reporter assay revealed that three theophylline riboswitches performed as expected in the cyanobacterium. Riboswitch-E* exhibited very low leaky expression of luciferase and superior and dose-dependent on/off regulation of protein expression by theophylline. The maximum magnitude of the induction vs. basal level was ∼190-fold. Furthermore, the induction level was responsive to a wide range of theophylline concentrations in the medium, from 0 to 2 mM, facilitating the fine-tuning of luciferase expression. We adapted this riboswitch to another gene regulation system, in which expression of the circadian clock kaiC gene product is controlled by the theophylline concentration in the culture medium. The results demonstrated that the adequately adjusted expression level of KaiC restored complete circadian rhythm in the kaiC-deficient arrhythmic mutant. This theophylline-dependent riboswitch system has potential for various applications as a useful genetic tool in cyanobacteria. © 2013 The Author.

DOI： 10.1093/pcp/pct115

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

A new response: A new type of hybridization switch-free ON-eRS (eukaryotic upregulating riboswitch) activates ribosomal shunting in response to a ligand. Whereas the ribosome that has finished sORF translation scans the 5′-side strand of the aptamer and stops at the rigid stem in the absence of the ligand, it shunts over the stem bearing the aptamer-ligand complex and then reinitiates translation of dORF in the presence of the ligand. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOI： 10.1002/cbic.201300362

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A sample preparation method was developed for single-nucleotide polymorphism (SNP) genotyping based on hybridization between a single-stranded DNA (ssDNA) analyte and an allele-specific oligonucleotide (ASO) probe. When the SNP site is located in the stable secondary structure, the folding of this analyte imposes kinetic penalties on the hybridization with the ASO probe. To address this issue, the sequence of the ssDNA analyte was converted from the original one so that the analyte exhibited a clear dumbbell-shaped structure composed of two stemloop moieties and an unfolded probe-binding site. The as-prepared analyte was structurally favorable for hybridization with the ASO probe, irrespective of the original sequence and secondary structure of the analyte. The sequence conversion was easily achieved by polymerase chain reaction using forward and reverse primers having an additional sequence at the 5'-terminus. These ssDNA analytes were subjected to affinity capillary electrophoresis using a diblock copolymer probe composed of an ASO segment and a poly(ethylene glycol) segment. The 70-base dumbbell-shaped analytes with a single-base difference were clearly separated within 12 min, although the original ones exhibited almost no separation due to the undesired folding of the probe-binding site. This sample preparation method should open up a wide range of applications for the ASO probes in genetic analysis.

DOI： 10.1021/ac400900b

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We report a facile design method for constructing multiple-input and visible-output logic gates by combining signal-converting DNA machines and the gold nanoparticle aggregation-based foundational AND gate.

DOI： 10.1039/c3ob40313k

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Here is presented a concept for in vitro selection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRN A Ser. Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRN A Ser. © 2012 Atsushi Ogawa et al.

DOI： 10.1155/2012/538129

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A strategy for rationally constructing a novel type of eukaryotic OFF-riboswitch, which ligand-dependently turns off translation mediated by an internal ribosome entry site (IRES), has been established. The theophylline-dependent IRES-based OFF-riboswitch obtained through the proposed strategy functioned well in wheat germ extract, independently from the downstream gene, indicating that it can regulate any gene. Despite the fact that it has one theophylline aptamer, its switching efficiency was as high as that of a previously reported theophylline-dependent OFF-riboswitch that was constructed by inserting three continuous theophylline aptamers into a 5' untranslated region in mRNA to downregulate the normal 5'-terminus-mediated translation. In addition, because the riboswitch part that was optimized in the theophylline-dependent IRES-based OFF-riboswitch, except for the aptamer domain, can be used as-is for other aptamer-ligand pairs, an arbitrary ligand-dependent IRES-based OFF-riboswitch is easy to construct with the corresponding well-minimized aptamer. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2011.12.118

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/cbic.201000744

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT  

Riboswitches are RNA elements in mRNA that control gene expression in cis in response to their specific ligands. Because artificial riboswitches make it possible to regulate any gene with an arbitrary molecule, they are expected to function as biosensors, in which the output is easily detectable protein expression. I report herein a fully rational design strategy for artificially constructing novel riboswitches that work in a eukaryotic cell-free translation system (wheat germ extract). In these riboswitches, translation mediated by an internal ribosome entry site (IRES) is promoted only in the presence of a specific ligand (ON), while it is inhibited in the absence of the ligand (OFF). The first rationally designed riboswitch, which is regulated by theophylline, showed a high switching efficiency and dependency on theophylline. In addition, based on the design of the theophylline-dependent riboswitch, other three kinds of riboswitches controlled by FMN, tetracycline, and sulforhodamine B, were constructed only by calculating the Delta G value of one stem-loop structure. The rational design strategy described herein is therefore useful for easily producing various ligand-dependent riboswitches, which are available as biosensors for detecting their ligands.

DOI： 10.1261/rna.2433111

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A single-step sensing system was developed to visually detect ligands of a cleavase-like RNA aptazyme at room temperature using aptazyme-tethered gold nanoparticles, the electrosteric stability of which was adjusted by increasing their diameter. In this system, the ligand induces self-cleavage of the aptazyme on gold nanoparticles to decrease the electrosteric stability of the gold nanoparticles, which causes them to visibly aggregate. In comparison to a previous multi-step system using aptazymes and gold nanoparticles separately, the present system requires only single handling and no special equipment, making it more suitable for on-the-spot sensing. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2010.11.048

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

DOI： 10.1039/c1ob06351k

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

An autonomous DNA machine recycling the output as the input for isothermal, sensitive, and specific detection of miRNAs has been developed. This machine shows considerably high signal amplification efficiency (similar to 1000-fold) and thus a low detection limit (similar to 20 amol). The machine also shows high specificity, discriminating 50 amol of synthetic miRNA from 100-fold larger amounts of its family member and from 100 ng of unrelated total RNAs. Moreover, it is available for practically detecting natural miRNAs in total RNAs. (c) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2010.08.055

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Language：English   Publishing type：Research paper (international conference proceedings)  

I have constructed a novel aptazyme-based biosensor system for detecting cofactors of the aptazymes using a cell-free luciferase synthesis in wheat germ extract. In this system, the activity of the aptazyme that is fused to a 5'-untranslated region of a luciferase gene can be detected as luciferase expression. In translating the aptazyme-fused mRNA as-is using a wheat germ cell-free translation system, the luciferase is almost not expressed because of the following triple suppression effects: (1) 5'-terminal three bases and (2) 5'-terminal duplex prevent the ribosome from binding to own mRNA; (3) if the ribosome binds, translation of a mimic gene in the aptazyme inhibits that of the downstream luciferase gene (OFF state). In contrast, in the presence of the aptazyme cofactor, the aptazyme in mRNA is self-cleaved to produce an aptazyme-free luciferase gene, which is translated efficiently (ON state). The ON/OFF efficiency and the detection limit of the aptazyme-based biosensor for theophylline are much higher and lower, respectively, compared to those of previously-reported one that utilizes a prokaryotic translation system.

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Language：English  

DOI： 10.1002/cbic.200900519

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

(Figure Presented) Sensible wheat germ: I have developed novel biosensors based on a new method for converting aptazyme activity into easily detectable protein expression by translating aptazyme-fused mRNA in eukaryotic wheat germ extract. Some newly elucidated unique features of the wheat germ translation system make the sensitivity of this method much higher than that of previously reported aptazyme-based biosensors. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/cbic.200900497

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：CHEMICAL SOC JAPAN  

We have developed a detector-free and multiple detection method for various analytes using some orthogonal pairs of cleavase-aptazymes and the corresponding probe-DNA-tethered gold nanoparticles (AuNPs) for each analyte. In this method, each cleavase-aptazyrne cleaves itself only in the presence of its cofactor (i.e. analyte), and each resulting cleaved RNA hybridizes only to the corresponding probe DNA on AuNPs to induce AuNP aggregation that can be detected with the naked eye.

DOI： 10.1246/cl.2009.848

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

We have developed an easy method for constructing aptazyme-based logic gates using noncrosslinking gold nanoparticle aggregation.

DOI： 10.1039/b910288d

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Language：English   Publishing type：Research paper (international conference proceedings)  

We developed a method for simply and rapidly detecting cofactors of aptazymes with high sensitivity using a unique noncrosslinking gold nanoparticle aggregation. Applying this method to a theophylline dependent aptazyme, 100 microM, 10 microM, and 1 microM theophylline were detected easily by the naked eye within 10 min, 20 min, and 65 min, respectively. This method is applicable to other cleavage-aptazymes without altering the probe-DNA sequence.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We developed a method for simply and rapidly detecting cofactors of aptazymes with high sensitivity using unique noncrosslinking gold nanoparticle aggregation. Applying this method to a theophylline-dependent aptazyme, 100 mu M, 10 mu M, and 1 mu M theophylline were detected easily by the naked eye within 10 min, 20 min, and 65 min, respectively. This method is also applicable to other cleavase-aptazymes without altering the probe-DNA sequence on the gold nanoparticle. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2008.10.051

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/cbic.200800294

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Authorship：Lead author,　Corresponding author   Language：Japanese  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

DOI： 10.1002/cbic.200700478

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (international conference proceedings)  

We constructed a new-type riboswitch, which functions in E. coli, using an aptazyme and an anti-RBS sequence. This riboswitch usually suppresses the gene expression with its anti-RBS sequence bound to the RBS of its own mRNA(OFF), while it activates the translation only when a cofactor of the aptazyme is added to release the anti-RBS sequence from itself as a result of cofactor-induced self-cleavage by the aptazyme (ON). Although this aptazyme-based riboswitch did not function at 37 degrees C in vivo in spite of its high activity at this temperature in vitro, it worked well at lower temperature (23 degrees C). We also improved the efficiency of this riboswitch by constructing a cascading system.

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We constructed a label-free and detector-free aptazyme-based riboswitch sensor for detecting the cofactor of the aptazyme. This riboswitch, which usually suppresses the gene expression with its anti-RBS sequence bound to the RBS of its own mRNA (OFF), activates the translation only when a cofactor is added to release the anti-RBS sequence from itself as a result of cofactor-induced self-cleavage by the aptazyme (ON). The rationally optimized one with beta-galactosidase as a reporter gene enabled us to detect the cofactor of the aptazyme visibly with high ON/OFF efficiency. (C) 2007 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2007.03.033

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A pool of 84-nt RNAs containing a randomized sequence of 50 nt was selected against gel-immobilized Escherichia coli release factor 1 (RF-1) responsible for translation termination at amber (UAG) stop codon. The strongest aptamer (class II-I) obtained from 43 clones bound to RF-1, but not to UAA/UGA-targeting RF-2, with K-d = 30 +/- 6 nM (SPR). A couple of impaired hairpin domains in the aptamer were suggested as the sites of attachment of RF-1. By binding to and hence inhibiting the action of RF-1 specifically or bio-orthogonally, aptamer class II-I enhanced the amber suppression efficiency in the presence of an anticodon-adjusted (CUA) suppressor tRNA without practically damaging the protein translation machinery of the cell-free extract of E coli, as confirmed by the translation of amber-mutated (gfp(amber141) or gfp(amber178)) and wild-type (gfp(wild)) genes of GFP. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.bmcl.2006.12.013

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Don't stop me now. Anticodon-adjusted tRNASer can be used as unified suppressors of the three types of stop codon. In their presence, translation of pseudonatural mRNAs for DHFR is rendered termination-free and proceeds into the untranslated region (UTR), giving rise to protein-ribosome- mRNA complex with full display of the protein when the translated UTR peptide serving as a spacer arm has a chain-length of ≥50 amino acids (aa). The mRNA template is recovered from the tag-selected complex with a 74-aa UTR in a yield of 2-4% based on input mRNA. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA.

DOI： 10.1002/cbic.200500397

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Authorship：Lead author   Language：English   Publishing type：Research paper (international conference proceedings)  

We carried out an in vitro selection of RNA aptamers that bind to Escherichia coli release factor 1 (E. coli RF1). The selected aptamer (class II) showed an apparent dissociation constant of nM range. The binding of the class II aptamer with E. coli RF1 is highly specific (orthogonal), allowing selective inhibition of RF1 activity in the E. coli translation system.

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Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

It has been experimentally established that the proton affinities (PA), as well as the solution basicities (pK(BH)(+)), of aziridine derivatives are much smaller than those of the corresponding pyrrolidines and piperidines, though the basic strength of azetidines is close to those of pyrrolidines and piperidines. A simple idea of dependence of the basic strength on bond angles seems to be invalid. Because the basicity of cyclic amines is a fundamental property in organic chemistry, we revisited this topic in order to clarify quantitatively the intrinsic origin of the strength of Lewis basicity of the relevant amines, in particular, based on the local electron-donating ability of the amine nitrogen atoms evaluated in terms of the localized reactive hybrid orbital (RHO) concept. In the cases of representative N-substituents such as hydrogen, methyl, and phenyl groups, the electron-donating energy level of the nitrogen center, obtained by maximizing a kind of superdelocalizability, was shown to be correlated with the magnitudes of experimental and calculated gas-phase proton affinities. The present results strongly support the view that the C-N-C bond angle, i.e., angle strain, in the cyclic amines is not the major source of the difference in strength of basicity of these amines, but rather, the degree of pyramidalization around the nitrogen atom has a significant impact on the electron-donating ability of the nitrogen lone-pair orbital.

DOI： 10.1021/jo0486589

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An affinity column immunobilizing a decapeptide H2N-RGDSPASSKP-CO2H was used to select RGD-binding aptamers from a pool of 86-mer single-strand oligodeoxynucleotides (ODNs) containing a random 40-mer sequence. The enriched library thus obtained was further selected against adsorbed fibronectin and individual aptamers were monocloned in E coli and sequenced to give a couple of highly homologous ODNs, which indeed inhibited fibronectin-integrin mediated cell adhesion. (C) 2004 Elsevier Ltd. All right, reserved.

DOI： 10.1016/j.bmcl.2004.05.042

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

C-Cu orbital interactions between a two-layer Cu-10 or three-layer Cu-34 cluster model of a Cu(111) surface and an adsorbed single C-60 molecule have been theoretically investigated, so as to elucidate the nature of the C-60-Cu(111) bonding and orientational configuration of the C-60 molecule on a Cu surface. Geometry optimizations and single-point calculations at the B3LYP/LanL2MB level of theory and fragment molecular orbital (FMO) analyses, coupled with a paired-interaction-orbital (PIO) scheme at the extended Hackel level of theory, have been performed for five symmetric adsorption models, in which a C-60 molecule is attached to the Cu-10 or Cu-34 cluster respectively by a six-membered ring (6-ring), by a five-membered ring (5-ring), by a C-C bond belonging to two 6-rings (6-6 bond), by a C-C bond belonging to a 6-ring and a 5-ring (5-6 bond), and by an edge carbon atom that is located at the center of two 6-rings and a 5-ring. Large stabilization is obtained for adsorption by an edge carbon atom or a 6-6 bond, whereas the other coordination types are not favored. Our result differs from an XPD experimental result for a C-60 monolayer on Cu(111), in which adsorption by a 6-ring is most favored. The discrepancy strongly suggests that C-60-C-60 interactions contribute significantly to the determination of C-60 orientations in C-60/Cu(111) monolayer systems.

DOI： 10.1021/jp0303220

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Recent studies predict that adenine radical cation (A•+) contributes to the hole-trapping process through long A/T sequences and exists as a real chemical intermediate. However, the experimental evidence for the existence of A•+ has not been observed in the DNA-mediated hole transport reaction. To examine the direct contribution of A•+, we have developed a novel hole-trapping nucleobase NA6-cyclopropyldeoxyadenosine (dCPA) which possesses a cyclopropyl group as a radical trapping device. One-electron oxidation of dCPA revealed that dCPA radical cation undergoes a rapid cyclopropane ring opening. With the use of the dCPA-containing DNA, we have demonstrated that the migrating hole was trapped at CPA incorporated into a long A/T bridge between two GG sites. The present results indicate that nucleobases possessing ionization potential higher than that of dG, such as dA, are able to participate directly in the multistep hopping mechanism. Copyright © 2003 American Chemical Society.

DOI： 10.1021/ja035887o

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

With the aim of understanding the nature of the S -Au(111) bonding in organosulfur/Au(111) self-assembled monolayer (SAM) systems, orbital interactions in the adsorption of methanethiolate (-SCH3) in various binding sites of a three-layer slab model and an Au-42 Cluster model of Au(111) surface are investigated. The methods of choice are crystal orbital overlap population (COOP) and crystal orbital Hamilton population (COHP) analyses for a periodic slab model and fragment molecular orbital (FMO) analyses for the cluster model. The origin of the S -Au(111) bond and the binding site preference are discussed from the viewpoint of orbital interaction. The site preference is in the order of three-fold hollow (fcc and hcp) > bridge > on-top. The second layer Au atoms have little influence on the S-Au(111) bonding, and adsorptions, to the fcc and hcp, sites are almost identical with respect to energy and S-Au bonding nature. Although sigma-type S-Au orbital interactions dominate the S-Au(111) bonding in the on-top model,pi-type S-Au orbital interactions play an important role in the bridge, fcc, and hcp models. FMO results,explain the vertical S-C bonds in the hollow models and the tilted S-C bonds in the on-top and bridge models.

DOI： 10.1021/jp020993i

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A reaction of 1,2-diketone with bis(iodozincio)methane gave a cyclopropanediol derivative via [2+1] cycloaddition. The reaction proceeded via a sequential nucleophilic attack of the dizinc reagent to a couple of carbonyl group in the substrate. The reaction proceeded with high diastereoselectivity to give cis-isomer. Detailed mechanistic studies have been examined by ab initio calculation. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0040-4020(02)00975-4

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

The Lewis acidity of GaF3, GaF2Cl, GaFCl2, and GaCl3 in acid-base interactions has been studied by taking ammonia as their electron-donating counterpart. We have derived an unoccupied reactive orbital that shows the maximum localization on the Ga atomic center for each species. The orbital is located lower in energy compared to those in the corresponding boron and aluminum halides. In contrast to boron halides, the unoccupied reactive orbital of the acid site tends to be delocalized considerably on the halogens as the fluorines are substituted by chlorines in gallium halides. The trend observed in the effects of fluorine and chlorine on the acidity of the gallium halides is opposite to those found in the boron halides. This cannot be interpreted solely in terms of the electron-accepting strength of the gallium center, but can be understood by including electrostatic interactions and closed-shell repulsion with ammonia in the adducts. The origin of the difference in Lewis acidity Of BCl3, AlCl3, and GaCl3 has been clarified.

DOI： 10.1021/ic020268m

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

An application of the perturbation theory to the [4+2] addition of butadiene has demonstrated that the effect of secondary orbital interactions should be much less significant than has been assumed within the frontier orbital scheme. This has been confirmed by a numerical analysis with respect to the endo transition state of the Diels-Alder reaction between butadiene and maleic anhydride. (C) 2002 Elsevier Science Ltd. All rights reserved.

DOI： 10.1016/S0040-4039(02)00164-8

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A guanine radical cation produced by one-electron DNA oxidation migrates over long distances through the DNA π-stack. Fundamental questions regarding the likelihood of charge transport in genomic DNA, the effects of protein binding, and its biological consequences arise as the next issues of study. Electronic effects of protein binding on the efficiency of charge transport were investigated for the endonuclease BamHI-DNA complex. Direct contact of a positively charged guanidium group of BamHI to guanines in the recognition sequence 5′-GGATCC-3′ completely suppressed one-electron oxidation of the guanine in the protein binding site and dramatically lowered the charge transport efficiency through the sequence. Electronically insulated guanines, by the hydrogen bonding contact of a guanidium group in BamHI, no longer function as a stepping stone in the charge transport through the DNA π-stack.

DOI： 10.1016/S1074-5521(02)00119-9

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Event date： 2021.3

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Event date： 2021.3

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Event date： 2020.11

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Event date： 2020.11

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Event date： 2020.10

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Event date： 2020.7

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Event date： 2020.5

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Language：Japanese  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：Japanese  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Presentation type：Oral presentation (general)  

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Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：English   Presentation type：Poster presentation  

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Applicant：国立大学法人京都大学

Application no：JP2005018680  Date applied：2005.10

Announcement no：WO2006-123445  Date announced：2006.11

WO 2006/123445

J-GLOBAL

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Applicant：国立大学法人京都大学

Application no：特願2004-057956  Date applied：2004.3

Announcement no：特開2005-245254  Date announced：2005.9

J-GLOBAL

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Applicant：独立行政法人科学技術振興機構

Application no：特願2001-072393  Date applied：2001.3

Announcement no：特開2002-275193  Date announced：2002.9

Patent/Registration no：特許第3681338号  Date issued：2005.5

J-GLOBAL

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Type：Promotional material

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Type：Research consultation

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Type：Seminar, workshop

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Type：University open house

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Type：University open house

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Type：University open house

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Type：University open house

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Type：University open house

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Type：University open house

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Type：University open house

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Type：University open house

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Type：Seminar, workshop

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Type：Seminar, workshop

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Type：Seminar, workshop

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Type：University open house

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Type：University open house

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