Papers - Doi, Nobuhide

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

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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

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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

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© 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

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© 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

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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