Details of a Researcher - Doi, Nobuhide

Papers - Doi, Nobuhide

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STALL-seq: mRNA-display selection of bacterial and eukaryotic translational arrest sequences from large random-sequence libraries

Hamano T., Nagumo Y., Umehara T., Hirono K., Fujiwara K., Taguchi H., Chadani Y., Doi N.

Journal of Biological Chemistry 300 ( 12 ) 107978 2024.12

ISSN 00219258

The translational arrest is a phenomenon wherein a temporary pause or slowing of the translation elongation reaction occurs due to the interaction between ribosome and nascent peptide. Recent studies have revealed that translational arrest peptides are involved in intracellular protein homeostasis regulatory functions, such as gene expression regulation at the translational level and regulation of cotranslational protein folding. Herein, we established a method for the large-scale in vitro selection of translational arrest peptides from DNA libraries by combining a modified mRNA display method and deep sequencing. We performed in vitro selection of translational arrest sequences from random-sequence libraries via mRNA display based on the Escherichia coli PURE system or wheat germ extract. Following several rounds of affinity selection, we obtained various candidate sequences that were not similar to known arrest peptides and subsequently confirmed their ribosome stalling activity by peptidyl-tRNA detection and toeprinting assay. Following the site-directed mutagenesis of the selected sequences, these clones were found to contain novel arrest peptide motifs. This method, termed STALL-seq (Selection of Translational Arrest sequences from Large Library sequencing), could be useful for the large-scale investigation of translational arrest sequences acting on both bacterial and eukaryotic ribosomes and could help discover novel intracellular regulatory mechanisms.

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Lipid nanoparticle-encapsulated DOCK11-siRNA efficiently reduces hepatitis B virus cccDNA level in infected mice

Okada H., Sakamoto T., Nio K., Li Y., Kuroki K., Sugimoto S., Shimakami T., Doi N., Honda M., Seiki M., Kaneko S., Yamashita T.

Molecular Therapy Methods and Clinical Development 32 ( 3 ) 101289 2024.09

ISSN 2329-0501

The hepatitis B virus (HBV) infects many people worldwide. As HBV infection frequently leads to liver fibrosis and carcinogenesis, developing anti-HBV therapeutic drugs is urgent. Therapeutic drugs for preventing covalently closed circular DNA (cccDNA) production, which can eliminate HBV infection, are unavailable. The host factor dedicator of cytokinesis 11 (DOCK11) is involved in the synthesis and maintenance of HBV cccDNA in vitro. However, the effectiveness of DOCK11 as a target for the in vivo elimination of HBV cccDNA remains unclear. In this study, we assess whether DOCK11 inhibitors suppress HBV cccDNA production in mouse models of HBV infection. The tocopherol-conjugate hetero- gapmer, a DNA/RNA duplex of gapmer/complementary RNA targeting the DOCK11 sequence, partially reduces the expression of DOCK11, but not that of HBV cccDNA, in the livers of HBV-infected human hepatocyte chimeric mice, along with weight loss and decreased serum human albumin levels. Lipid nanoparticle-encapsulated chemically modified siRNAs specific for DOCK11 suppress DOCK11 expression and decrease HBV cccDNA levels without adverse effects in the mice. Therefore, nucleic acid-based drugs targeting DOCK11 in hepatocytes are potentially effective anti-HBV therapeutics that can reduce HBV cccDNA levels in vivo.

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Cell-Free Protein Expression by a Reconstituted Transcription–Translation System Energized by Sugar Catabolism

Sato G., Miyazawa S., Doi N., Fujiwara K.

Molecules 29 ( 13 ) 2024.07

Cooperation between catabolism and anabolism is crucial for maintaining homeostasis in living cells. The most fundamental systems for catabolism and anabolism are the glycolysis of sugars and the transcription–translation (TX-TL) of DNA, respectively. Despite their importance in living cells, the in vitro reconstitution of their cooperation through purified factors has not been achieved, which hinders the elucidation of the design principle in living cells. Here, we reconstituted glycolysis using sugars and integrated it with the PURE system, a commercial in vitro TX-TL kit composed of purified factors. By optimizing key parameters, such as glucokinase and initial phosphate concentrations, we determined suitable conditions for their cooperation. The optimized system showed protein synthesis at up to 33% of that of the original PURE system. We observed that ATP consumption in upstream glycolysis inhibits TX-TL and that this inhibition can be alleviated by the co-addition of glycolytic intermediates, such as glyceraldehyde 3-phosphate, with glucose. Moreover, the system developed here simultaneously synthesizes a subset of its own enzymes, that is, glycolytic enzymes, in a single test tube, which is a necessary step toward self-replication. As glycolysis and TX-TL provide building blocks for constructing cells, the integrated system can be a fundamental material for reconstituting living cells from purified factors.

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Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution

Sato G., Kinoshita S., Yamada T.G., Arai S., Kitaguchi T., Funahashi A., Doi N., Fujiwara K.

ACS Synthetic Biology 13 ( 5 ) 1572 - 1581 2024.05

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

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Multimolecular Competition Effect as a Modulator of Protein Localization and Biochemical Networks in Cell-Size Space

Nishikawa S., Sato G., Takada S., Kohyama S., Honda G., Yanagisawa M., Hori Y., Doi N., Yoshinaga N., Fujiwara K.

Advanced Science 11 ( 6 ) e2308030 2024.02

Cells are small, closed spaces filled with various types of macromolecules. Although it is shown that the characteristics of biochemical reactions in vitro are quite different from those in living cells, the role of the co-existence of various macromolecules in cell-size space remains still elusive. Here, using a constructive approach, it is demonstrated that the co-existence of various macromolecules themselves has the ability to tune protein localization for spatiotemporal regulation and a biochemical reaction system in a cell-size space. Both experimental and theoretical analyses reveal that enhancement of interfacial effects by a large surface-area-to-volume ratio facilitates membrane localization of molecules in the cell-size space, and the interfacial effects are alleviated by competitive binding to lipid membranes among multiple proteins even if their membrane affinities are weak. These results indicate that competition for membrane binding among various macromolecules in the cell-size space plays a role in regulating the spatiotemporal molecular organization and biochemical reaction networks. These findings shed light on the importance of surrounding molecules for biochemical reactions using purified elements in small spaces.

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Cytoplasmic delivery of siRNA using human-derived membrane penetration-enhancing peptide

Nakamura M., Fujiwara K., Doi N.

Journal of Nanobiotechnology 20 ( 1 ) 458 2022.12

Background: Although protein-based methods using cell-penetrating peptides such as TAT have been expected to provide an alternative approach to siRNA delivery, the low efficiency of endosomal escape of siRNA/protein complexes taken up into cells by endocytosis remains a problem. Here, to overcome this problem, we adopted the membrane penetration-enhancing peptide S19 from human syncytin 1 previously identified in our laboratory. Results: We prepared fusion proteins in which the S19 and TAT peptides were fused to the viral RNA-binding domains (RBDs) as carrier proteins, added the RBD-S19-TAT/siRNA complex to human cultured cells, and investigated the cytoplasmic delivery of the complex and the knockdown efficiency of target genes. We found that the intracellular uptake of the RBD-S19-TAT/siRNA complex was increased compared to that of the RBD-TAT/siRNA complex, and the expression level of the target mRNA was decreased. Because siRNA must dissociate from RBD and bind to Argonaute 2 (Ago2) to form the RNA-induced silencing complex (RISC) after the protein/siRNA complex is delivered into the cytoplasm, a dilemma arises: stronger binding between RBD and siRNA increases intracellular uptake but makes RISC formation more difficult. Thus, we next prepared fusion proteins in which the S19 and TAT peptides were fused with Ago2 instead of RBD and found that the efficiencies of siRNA delivery and knockdown obtained using TAT-S19-Ago2 were higher than those using TAT-Ago2. In addition, we found that the smallest RISC delivery induced faster knockdown than traditional siRNA lipofection, probably due to the decreased time required for RISC formation in the cytoplasm. Conclusion: These results indicated that S19 and TAT-fused siRNA-binding proteins, especially Ago2, should be useful for the rapid and efficient delivery of siRNA without the addition of any endosome-disrupting agent.

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Controlling the Periodicity of a Reaction-Diffusion Wave in Artificial Cells by a Two-Way Energy Supplier

Takada S., Yoshinaga N., Doi N., Fujiwara K.

ACS Nano 16 ( 10 ) 16853 - 16861 2022.10

ISSN 19360851

Reaction-diffusion (RD) waves, which are dynamic self-organization structures generated by nanosize molecules, are a fundamental mechanism from patterning in nano- and micromaterials to spatiotemporal regulations in living cells, such as cell division and motility. Although the periods of RD waves are the critical element for these functions, the development of a system to control their period is challenging because RD waves result from nonlinear physical dynamics under far-from-equilibrium conditions. Here, we developed an artificial cell system with tunable period of an RD-driven wave (Min protein wave), which determines a cell division site plane in living bacterial cells. The developed system is based on our finding that Min waves are generated by energy consumption of either ATP or dATP, and the period of the wave is different between these two energy suppliers. We showed that the Min-wave period was modulated linearly by the mixing ratio of ATP and dATP and that it was also possible to estimate the mixing ratio of ATP and dATP from the period. Our findings illuminated a previously unidentified principle to control the dissipative dynamics of biomolecules and, simultaneously, built an important framework to construct molecular robots with spatiotemporal units.

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Mode selection mechanism in traveling and standing waves revealed by Min wave reconstituted in artificial cells

Takada S., Yoshinaga N., Doi N., Fujiwara K.

Science advances (Science advances) 8 ( 23 ) eabm8460 2022.06

Reaction-diffusion coupling (RDc) generates spatiotemporal patterns, including two dynamic wave modes: traveling and standing waves. Although mode selection plays a substantial role in the spatiotemporal organization of living cell molecules, the mechanism for selecting each wave mode remains elusive. Here, we investigated a wave mode selection mechanism using Min waves reconstituted in artificial cells, emerged by the RDc of MinD and MinE. Our experiments and theoretical analysis revealed that the balance of membrane binding and dissociation from the membrane of MinD determines the mode selection of the Min wave. We successfully demonstrated that the transition of the wave modes can be regulated by controlling this balance and found hysteresis characteristics in the wave mode transition. These findings highlight a previously unidentified role of the balance between activators and inhibitors as a determinant of the mode selection of waves by RDc and depict an unexplored mechanism in intracellular spatiotemporal pattern formations.

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Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits Hepatitis B Virus infection and replication.

Ide M, Tabata N, Yonemura Y, Shirasaki T, Murai K, Wang Y, Ishida A, Okada H, Honda M, Kaneko S, Doi N, Ito S, Yanagawa H

The Journal of biological chemistry 298 ( 7 ) 102097 2022.06

ISSN 0021-9258

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

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Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins

Suzuki M., Iwaki K., Kikuchi M., Fujiwara K., Doi N.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 586 63 - 67 2022.01

ISSN 0006291X

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high β-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the β-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a β-strand and that the subsequent parallel β-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

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Activation of a diluted E. coli cell-free transcription-translation system within liposomes by hypertonic concentration

Matsui Y., Akui T., Doi N., Fujiwara K.

STAR Protocols (STAR Protocols) 2 ( 4 ) 101003 2021.12

We present a protocol for activating protein synthesis in liposomes encapsulating a diluted E. coli cell extract-based TX-TL (transcription-translation) system by hypertonic concentration. Protein expression is turned on in the liposome-encapsulated TX-TL system by simple treatment with a concentrated external solution. The expression of sfGFP is demonstrated here, but it can be applied to other proteins. This protocol can be applied to the development of artificial cells utilizing the switch-on mechanism to activate protein expression, responding to the outer environment. For complete details on the use and execution of this protocol, please refer to Akui et al. (2021).

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Transcription-translation of theEscherichia coligenome within artificial cells

Deyama T., Matsui Y., Chadani Y., Sekine Y., Doi N., Fujiwara K.

Chemical Communications (Chemical Communications) 57 ( 80 ) 10367 - 10370 2021.10

ISSN 13597345

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System concentration shift as a regulator of transcription-translation system within liposomes

Akui T., Fujiwara K., Sato G., Takinoue M., Nomura S.i.M., Doi N.

iScience (iScience) 24 ( 8 ) 102859 2021.08

Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

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Switching ON of Transcription-Translation System Using GUV Fusion by Co-supplementation of Calcium with Long-Chain Polyethylene Glycol

Uwaguchi Y., Fujiwara K., Doi N.

ChemBioChem (ChemBioChem) 22 ( 13 ) 2319 - 2324 2021.07

ISSN 14394227

Giant unilamellar vesicles (GUVs) have been used as a material for bottom-up synthetic biology. However, due to the semi-permeability of the membrane, the need for methods to fuse GUVs has increased. To this aim, methods that are simple and show low leakage during fusion are important. In this study, we report a method of GUV fusion by a divalent cation (Ca2+) enhanced with a long chain polyethylene glycol (PEG20k). The methods showed significant GUV fusion without leakage of internal components of GUVs and maintained cell-free transcription-translation ability inside the GUVs without external supplementation of macromolecules. We demonstrate that the Ca-PEG method can be applied for switching ON of transcription-translation in GUVs in a fusion-dependent manner. The method developed here can be applied to extend bottom-up synthetic biology and molecular robotics that use GUVs as a chassis.

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A relationship between ntp and cell extract concentration for cell-free protein expression

Takahashi K., Sato G., Doi N., Fujiwara K.

Life (Life) 11 ( 3 ) 2021.03

ISSN 2075-1729

The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

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Conformational equilibrium of MinE regulates the allowable concentration ranges of a protein wave for cell division

Kohyama S., Fujiwara K., Yoshinaga N., Doi N.

Nanoscale (Nanoscale) 12 ( 22 ) 11960 - 11970 2020.06

ISSN 20403364

The Min system for determining the cell division position at the center in bacteria has a unique character that uses a protein wave (Min wave) that emerges from its components (MinD and MinE). The Min wave emerges under the coupling of chemical reactions and molecular diffusions of MinDE and appears when the concentrations of MinD and MinE are similar. However, the nanoscale mechanism to determine their concentration ranges has remained elusive. In this study, by using artificial cells as a mimic of cells, we showed that the dominant MinE conformations determined the allowable concentration ranges for the emergence of the Min wave. Furthermore, the deletion of the membrane-binding region of MinE indicated that the region was essential for limiting the concentration ranges to be narrower. These findings illustrate a parameter tuning mechanism underlying complex molecular systems at the nanoscale for spatiotemporal regulation in living cells and show a possibility that the regulation of the equilibrium among molecular conformations can work as a switch for cell division.

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Self-organization Assay for Min Proteins of <i>Escherichia coli</i> in Micro-droplets Covered with Lipids.

Kohyama S, Fujiwara K, Yoshinaga N, Doi N

Bio-protocol 10 ( 6 ) e3561 2020.03

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Microfluidic screening system based on boron-doped diamond electrodes and dielectrophoretic sorting for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Oyobiki R., Fujiwara K., Watanabe T., Einaga Y., Matsumoto Y., Miki N., Doi N.

Lab on a Chip (Lab on a Chip) 20 ( 4 ) 852 - 861 2020.02

ISSN 14730197

We report the development of a micro total analysis system (μTAS) based on electrochemical measurements and dielectrophoretic sorting for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzymes immobilized on microbeads, together with their encoding DNA, can be measured with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. The 30 nL droplets containing microbead-displayed genes of enzymes with higher activity can then be recovered by dielectrophoretic sorting. Previously, we developed the NAD(P)H-measuring device containing the BDD electrode for high-throughput measurement of the activity of NAD(P)-dependent oxidoreductases. In this study, we fabricated an encapsulating device and a droplet-sorting device for nanoliter-size droplets, for the first time, and then combined these three devices to construct a μTAS for directed evolution of NAD(P)-dependent oxidoreductases. We confirmed that this system works by proof-of-principle experiments and successfully applied this system for screening of randomized libraries of NAD-dependent dehydrogenases.

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Dielectrophoretic sorting system for screening of NAD(P)-dependent oxidoreductases.

Kanai Y., Shimokihara S., Shitara S., Oyobiki R., Watanabe T., Einaga Y., Wakamatsu K., Matsumoto Y., Fujiwara K., Doi N.

21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017 (21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2017) 1383 - 1384 2020

Here we report the development of a flow injection analysis system based on electrochemical measurements and dielectrophoresis for screening of NAD(P)-dependent oxidoreductases. In this system, the activity of enzyme which is immobilized on microbeads together with its encoding DNA can be measured with a boron-doped diamond (BDD) electrode in each compartment (~30 nL) of the microfluidic system, and the large droplets (~30 nL) containing genes of enzymes with higher activity can be recovered by dielectrophoretic sorting.

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells

Kohyama S., Yoshinaga N., Yanagisawa M., Fujiwara K., Doi N.

eLife (eLife) 8 2019.07

© Kohyama et al. The Min system, a system that determines the bacterial cell division plane, uses changes in the localization of proteins (a Min wave) that emerges by reaction-diffusion coupling. Although previous studies have shown that space sizes and boundaries modulate the shape and speed of Min waves, their effects on wave emergence were still elusive. Here, by using a microsized fully confined space to mimic live cells, we revealed that confinement changes the conditions for the emergence of Min waves. In the microsized space, an increased surface-tovolume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary for the stable generation of Min waves. Furthermore, we showed that the cell-sized space strictly limits parameters for wave emergence because confinement inhibits both the instability and excitability of the system. These results show that confinement of reaction-diffusion systems has the potential to control spatiotemporal patterns in live cells.

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Regulation of spatiotemporal patterning in artificial cells by a defined protein expression system

Yoshida A., Kohyama S., Fujiwara K., Nishikawa S., Doi N.

Chemical Science (Chemical Science) 10 ( 48 ) 11064 - 11072 2019

ISSN 20416520

Spatiotemporal patterning is a fundamental mechanism for developmental differentiation and homeostasis in living cells. Because spatiotemporal patterns are based on higher-order collective motions of elements synthesized from genes, their behavior dynamically changes according to the element amounts. Thus, to understand life and use this process for material application, creation of artificial cells with time development of spatiotemporal patterning by changes of element levels is necessary. However, realizing coupling between spatiotemporal patterning and synthesis of elements in artificial cells has been particularly challenging. In this study, we established a system that can synthesize a patterning mechanism of the bacterial cell division plane (the so-called Min system) in artificial cells by modifying a defined protein expression system and demonstrated that artificial cells can show time development of spatiotemporal patterning similar to living cells. This system also allows generation and disappearance of spatiotemporal patterning, is controllable by a small molecule in artificial cells, and has the ability for application in cargo transporters. The system developed here provides a new material and a technique for understanding life, development of drug delivery tools, and creation of molecular robots.

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A dual system using compartmentalized partnered replication for selection of arsenic-responsive transcriptional regulator

Aye S., Fujiwara K., Doi N.

J. Biochem. (Journal of Biochemistry) 164 ( 5 ) 341 - 348 2018.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021924X

Engineering and design of genetic circuit in living cell is critical in accessing the beneficial application of synthetic biology. Directed evolution can avoid the complicated rational design of such circuit by screening or selecting functional circuit from non-functional one. Here, we proposed a positive-negative selection system for selecting a transcription factor that activates gene expression in response to arsenic in solution. First, we developed a whole cell biosensor for sensing arsenite in liquid using a regulator (ArsR) and a reporter (GFP), and evaluated its performance. Second, we developed a positive selection system for active ArsR using compartmentalized partnered replication that uses thermostable DNA polymerase as the reporter of activity. Third, we developed a negative selection system using sucrose-induced suicide gene SacB as the reporter for exclusion of inactive ArsR variants.

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Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Aye, S.L., Fujiwara, K., Ueki, A., Doi, N.

Biochem. Biophys. Res. Commun. (Biochemical and Biophysical Research Communications) 499 ( 2 ) 170 - 176 2018.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0006291X

© 2018 Elsevier Inc. Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

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Artificial cell fermentation as a platform for highly efficient cascade conversion

Fujiwara, K., Adachi, T., Doi, N.

ACS Synth. Biol. (ACS Synthetic Biology) 7 ( 2 ) 363 - 370 2018.02

Research paper (scientific journal), Joint Work, Accepted

© 2017 American Chemical Society. Because of its high specificity and stereoselectivity, cascade reactions using enzymes have been attracting attention as a platform for chemical synthesis. However, the sensitivity of enzymes outside their optimum conditions and their rapid decrease of activity upon dilution are drawbacks of the system. In this study, we developed a system for cascade enzymatic conversion in bacteria-shaped liposomes formed by hypertonic treatment, and demonstrated that the system can overcome the drawbacks of the enzymatic cascade reactions in bulk. This system produced final products at a level equivalent to the maximum concentration of the bulk system (0.10 M, e.g., 4.6 g/L), and worked even under conditions where enzymes normally lose their function. Under diluted conditions, the conversion rate of the artificial cell system was remarkably higher than that in the bulk system. Our results indicate that artificial cells can behave as a platform to perform fermentative production like microorganisms.

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Improvement of microtas for directed evolution of NAD(P)-dependent oxidoreductases

Goto H., Kanai Y., Yotsui A., Shimokihara S., Shitara S., Einaga Y., Matsumoto Y., Miki N., Fujiwara K., Doi N.

22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018 (22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018) 4 2247 - 2249 2018

Here we report an improved μTAS based on electrochemical measurements and microbeads sorting for screening of NAD(P)-dependent oxidoreductases. By using this system, we measured the activity of model enzymes immobilized on microbeads by detection of NADH with a boron-doped diamond (BDD) electrode in each compartment (∼30 nL) of the microfluidic system. Then, we recovered the magnetic microbeads or the droplets containing genes of enzymes with higher activity by magnetophoretic or dielectrophoretic sorting, respectively. Our μTAS should be useful for directed evolution of various NAD(P)-dependent oxidoreductases with high activity and stability in the future.

In vitro transcription–translation using bacterial genome as a template to reconstitute intracellular profile

Fujiwara, K., Sawamura, T., Niwa, T., Deyama, T., Nomura, S.M., Taguchi, H., Doi, N.

Nucleic Acids Res. 45 ( 19 ) 11449 - 11458 2017.11

Research paper (scientific journal), Joint Work, Accepted

Human-derived fusogenic peptides for the intracellular delivery of proteins

Sudo, K., Niikura, K., Iwaki, K., Kohyama, S., Fujiwara, K., Doi, N.

J. Control. Release 255 1 - 11 2017.04

Research paper (scientific journal), Joint Work, Accepted

In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display

Nakayama, M., Komiya, S., Fujiwara, K., Horisawa, K., Doi, N.

Biochem. Biophys. Res. Commun. 478 ( 2 ) 606 - 611 2016.09

Research paper (scientific journal), Joint Work, Accepted

PURE mRNA display for in vitro selection of single-chain antibodies

Nagumo, Y., Fujiwara, K., Horisawa, K., Yanagawa, H., Doi, N.

J. Biochem. 159 ( 5 ) 519 - 526 2016.05

Research paper (scientific journal), Joint Work, Accepted

Biochemical preparation of cell extract for cell-free protein synthesis without physical disruption

Fujiwara, K., Doi, N.

PLoS ONE 11 ( 10 ) e0154614 2016.04

Research paper (scientific journal), Joint Work, Accepted

Endosomal escape efficiency of fusogenic B18 and B55 peptides fused with anti-EGFR single chain Fv as estimated by nuclear translocation

Niikura, K., Horisawa, K., Doi, N.

J. Biochem. 159 123 - 132 2015.09

Research paper (scientific journal), Joint Work, Accepted

A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape

Niikura, K., Horisawa, K., Doi, N.

J. Control. Release 212 85 - 93 2015.08

Research paper (scientific journal), Joint Work, Accepted

Toward high-throughput screening of NAD(P)-dependent oxidoreductases using boron-doped diamond microelectrodes and microfluidic devices

Oyobiki, R., Kato, T., Katayama, M., Sugitani, A., Watanabe, T., Einaga, Y., Matsumoto, Y., Horisawa, K., Doi, N.

Anal. Chem. 86 ( 19 ) 9570 - 9575 2014.10

Research paper (scientific journal), Joint Work, Accepted

Structural basis for inhibition of the MDM2:p53 interaction by an optimized MDM2-binding peptide selected with mRNA display

Nagata, T., Shirakawa, K., Kobayashi, N., Shiheido, H., Tabata, N., Sakuma-Yonemura, Y., Horisawa, K., Katahira, M., Doi, N., Yanagawa, H.

PLoS ONE 9 ( 10 ) e109163 2014.10

Research paper (scientific journal), Joint Work, Accepted

MIP-2A is a novel target of an anilinoquinazoline derivative for inhibition of tumor cell proliferation

Tokunaga, M., Shiheido, H., Tabata, N., Sakuma-Yonemura, Y., Takashima, H., Horisawa, K., Doi, N., Yanagawa, H.

PLoS ONE 8 ( 9 ) e76774 2013.09

Research paper (scientific journal), Joint Work, Accepted

Hereditary spastic paraplegia protein spartin is an FK506-binding protein identified by mRNA display

Tokunaga, M., Shiheido, H., Hayakawa, I., Utsumi, A., Takashima, H., Doi, N., Horisawa, K., Sakuma-Yonemura, Y., Tabata, N., Yanagawa, H.

Chem. Biol. 20 ( 7 ) 935 - 942 2013.07

Research paper (scientific journal), Joint Work, Accepted

In vitro selection of Fab fragments by mRNA display and gene-linking emulsion PCR

Sumida, T., Yanagawa, H., Doi, N.

J. Nucleic Acids 2012 371379 2012.09

Research paper (scientific journal), Joint Work, Accepted

An anilinoquinazoline derivative inhibits tumor growth through interaction with hCAP-G2, a subunit of condensin II

Shiheido, H., Naito, Y., Kimura, H., Genma, H., Takashima, H., Tokunaga, M., Ono, T., Hirano, T., Du, W., Yamada, T., Doi, N., Iijima, S., Hattori, Y., Yanagawa, H.

PLoS ONE 7 ( 9 ) e44889 2012.09

Research paper (scientific journal), Joint Work, Accepted

A phthalimide derivative that inhibits centrosomal clustering is effective on multiple myeloma

Shiheido, H., Terada, F., Tabata, N., Hayakawa, I., Matsumura, N., Takashima, H., Ogawa, Y., Du, W., Yamada, T., Shoji, M., Sugai, T., Doi, N., Iijima, S., Hattori, Y., Yanagawa, H.

PLoS ONE 7 ( 6 ) e38878 2012.06

Research paper (scientific journal), Joint Work, Accepted

DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells

Doi, N., Yamakawa, N., Matsumoto, H., Yamamoto, Y., Nagano, T., Matsumura, N., Horisawa, K., Yanagawa, H.

PLoS ONE 7 ( 1 ) e30084 2012.01

Research paper (scientific journal), Joint Work, Accepted

Comparison of the frequency of functional SH3 domains with different limited sets of amino acids using mRNA display

Tanaka, J., Yanagawa, H., Doi, N.

PLoS ONE 6 ( 3 ) e18034 2011.03

Research paper (scientific journal), Joint Work, Accepted

mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction

Shiheido, H., Takashima, H., Doi, N., Yanagawa, H.

PLoS ONE 6 ( 3 ) e17898 2011.03

Research paper (scientific journal), Joint Work, Accepted

Electrochemical detection of tyrosine derivatives and protein tyrosine kinase activity using boron-doped diamond electrodes

Chiku, M., Horisawa, K., Doi, N., Yanagawa, H., Einaga, Y.

Biosens. Bioelectron. 26 ( 1 ) 235-240 2010.09

Research paper (scientific journal), Joint Work, Accepted

mRNA display selection of a high-affinity, Bcl-XL-specific binding peptide

Matsumura, N., Tsuji, T., Sumida, T., Kokubo, M., Onimaru, M., Doi, N., Takashima, H., Miyamoto-Sato, E., Yanagawa, H.

FASEB J. 24 ( 7 ) 2201-2210 2010.07

Research paper (scientific journal), Joint Work, Accepted

Comparative characterization of random-sequence proteins consisting of 5, 12 and 20 kinds of amino acids

Tanaka, J., Doi, N., Takashima, H., Yanagawa, H.

Protein Sci. 19 ( 4 ) 786-795 2010.04

Research paper (scientific journal), Joint Work, Accepted

In vitro selection of GTP-binding proteins by block shuffling of estrogen-receptor fragments

Tsuji, T., Onimaru, M., Doi, N., Miyamoto-Sato, E., Takashima, H., Yanagawa, H.

Biochem. Biophys. Res. Commun. 390 ( 3 ) 689-693 2009.10

Research paper (scientific journal), Joint Work, Accepted

Bicistronic DNA display for in vitro selection of Fab fragments

Sumida, T., Doi, N., Yanagawa, H.

Nucleic Acids Res. 37 ( 22 ) e147 2009.09

Research paper (scientific journal), Joint Work, Accepted

Rapid antibody selection by mRNA display on a microfluidic chip

Tabata, N., Sakuma, Y., Honda, Y., Doi, N., Takashima, H., Miyamoto-Sato, E., Yanagawa, H.

Nucleic Acids Res. 37 ( 8 ) e64 2009.03

Research paper (scientific journal), Joint Work, Accepted

Use of cDNA tiling arrays for identifying protein-interactions selected by in vitro display technologies

Horisawa, K., Doi, N., Yanagawa, H.

PLoS ONE 3 ( 2 ) e1646 2008.02

Research paper (scientific journal), Joint Work, Accepted

Photocleavable linkage between genotype and phenotype for rapid and efficient recovery of nucleic acids encoding affinity-selected proteins.

Doi, N., Takashima, H., Wada, A., Oishi, Y., Nagano, T., Yanagawa, H.

J. Biotechnol. 131 ( 3 ) 231-239 2007.08

Research paper (scientific journal), Joint Work, Accepted

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis (OGAB).

Nishizaki, T., Tsuge, K., Itaya, M., Doi, N., Yanagawa, H.

Appl. Environ. Microbiol. 73 ( 4 ) 1355-1361 2007.02

Research paper (scientific journal), Joint Work, Accepted

High-throughput fluorescence labeling of full-length cDNA products based on a reconstituted translation system

Kawahashi, Y., Doi, N., Oishi, Y., Tsuda, C., Takashima, H., Baba, T., Mori, H., Ito, T., Yanagawa, H.

J. Biochem. 141 ( 1 ) 19-24 2007.01

Research paper (scientific journal), Joint Work, Accepted

Escherichia coli proteome chips for detecting protein-protein interactions

Oishi, Y., Yunomura, S., Kawahashi, Y., Doi, N., Takashima, H., Baba, T., Mori, H., Yanagawa, H.

Proteomics 6 ( 24 ) 6433-6436 2006.12

Research paper (scientific journal), Joint Work, Accepted

Recent Progress and Future Prospects in Protein Display Technologies as Tools for Proteomics

Matsumura, N., Doi, N., Yanagawa, H.

Current Proteomics 3 ( 3 ) 199-215 2006.10

Research paper (scientific journal), Joint Work, Accepted

In vitro evolution of single-chain antibodies using mRNA display

Fukuda, I., Kojoh, K., Tabata, N., Doi, N., Takashima, H., Miyamoto-Sato, E., Yanagawa, H.

Nucleic Acids Res. 34 ( 19 ) e127 2006.09

Research paper (scientific journal), Joint Work, Accepted

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Oyama, R., Takashima, H., Yonezawa, M., Doi, N., Miyamoto-Sato, E., Kinjo, M., Yanagawa, H.

Nucleic Acids Res. 34 ( 14 ) e102 2006.08

Research paper (scientific journal), Joint Work, Accepted

Affinity selection of DNA-binding protein complexes using mRNA display

Tateyama, S., Horisawa, K., Takashima, H., Miyamoto-Sato, E., Doi, N., Yanagawa, H.

Nucleic Acids Res. 34 ( 3 ) e27 2006.02

Research paper (scientific journal), Joint Work, Accepted

High solubility of random-sequence proteins consisting of five kinds of primitive amino acids

Doi, N., Kakukawa, K., Oishi, Y., Yanagawa, H.

Protein Engineering, Design & Selection 18 ( 6 ) 279-284 2005.05

Research paper (scientific journal), Joint Work, Accepted

Application of quantitative real-time PCR for monitoring the process of enrichment of clones on in vitro protein selection

Horisawa, K., Doi, N., Takashima, H., Yanagawa, H.

J. Biochem. 137 ( 2 ) 121-124 2005.02

Research paper (scientific journal), Joint Work, Accepted

Inversion of enantioselectivity of asymmetric biocatalytic decarboxylation by site-directed mutagenesis based on the reaction mechanism

18. Ijima, Y., Matoishi, K., Terao, Y., Horisawa, K., Doi, N., Takashima, H., Yanagawa, H., Ohta, H.

Chem. Commun. ( 7 ) 877-879 2005.01

Research paper (scientific journal), Joint Work, Accepted

In vitro selection of Jun-associated proteins using mRNA display

Horisawa, K., Tateyama, S., Ishizaka, M., Matsumura, N., Takashima, H., Miyamoto-Sato, E., Doi, N., Yanagawa, H.

Nucleic Acids Res. 32 ( 21 ) e169 2004.12

Research paper (scientific journal), Joint Work, Accepted

In vitro selection of restriction endonucleases by in vitro compartmentalization

Doi, N., Kumadaki, S., Oishi, Y., Matsumura, N., Yanagawa, H.

Nucleic Acids Res. 32 ( 12 ) e95 2004.07

Research paper (scientific journal), Joint Work, Accepted

DNA display of biologically active proteins for in vitro protein selection

Yonezawa, M., Doi, N., Higashinakagawa, T., Yanagawa, H.

J. Biochem. 135 ( 3 ) 285-288 2004.04

Research paper (scientific journal), Joint Work, Accepted

DNA display for in vitro selection of diverse peptide libraries

Yonezawa, M., Doi, N., Kawahashi, Y., Higashinakagawa, T., Yanagawa, H.

Nucleic Acids Res. 31 ( 19 ) E118 2003.10

Research paper (scientific journal), Joint Work, Accepted

In vitro protein microarrays for detecting protein-protein interactions: Application of a new method for fluorescence labeling of proteins

Kawahashi, Y., Doi, N., Takashima, H., Tsuda, C., Oishi, Y., Oyama, R., Yonezawa, M., Miyamoto-Sato, E., Yanagawa, H.

Proteomics 3 1236-1243 2003.07

Research paper (scientific journal), Joint Work, Accepted

Evolutionary design of generic GFP biosensors

Doi, N., Yanagawa, H.

Methods Mol. Biol. 183 49-55 2002.08

Research paper (scientific journal), Joint Work, Accepted

Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions

Doi, N., Takashima, H., Kinjo, M., Sakata, K., Kawahashi, Y., Oishi, Y., Oyama, R., Miyamoto-Sato, E., Sawasaki, T., Endo, Y., Yanagawa, H.

Genome Res. 12 487-492 2002.03

Research paper (scientific journal), Joint Work, Accepted

Genotype-phenotype linkage for directed evolution and screening of combinatorial protein libraries

Doi, N., Yanagawa, H.

Comb. Chem. High Throughput Screen. 4 497-509 2001.09

Research paper (scientific journal), Joint Work, Accepted

STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro

Doi, N., Yanagawa, H.

FEBS Lett. 457 227-230 1999.08

Research paper (scientific journal), Joint Work, Accepted

Insertional gene fusion technology

Doi, N., Yanagawa, H.

FEBS Lett. 457 1-4 1999.07

Research paper (scientific journal), Joint Work, Accepted

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution

Doi, N., Yanagawa, H.

FEBS Lett. 453 305-307 1999.06

Research paper (scientific journal), Joint Work, Accepted

Origins of globular structure in proteins

Doi, N., Yanagawa, H.

FEBS Lett. 430 150-153 1998.07

Research paper (scientific journal), Joint Work, Accepted

Screening of conformationally constrained random polypeptide libraries displayed on a protein scaffold

Doi, N., Yanagawa, H.

Cell. Mol. Life Sci. 54 394-404 1998.05

Research paper (scientific journal), Joint Work, Accepted

Characterization of random-sequence proteins displayed on the surface of Escherichia coli RNase HI

Doi, N., Yomo, T., Itaya, M., Yanagawa, H.

FEBS Lett. 427 51-54 1998.05

Research paper (scientific journal), Joint Work, Accepted

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme

Doi, N., Itaya, M., Yomo, T., Tokura, S., Yanagawa, H.

FEBS Lett. 402 177-180 1997.02

Research paper (scientific journal), Joint Work, Accepted

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