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Ueno, Shingo

Research contents (Research key words)

Proteins such as antibodies, peptides, and enzymes possess highly sophisticated functions and find broad applications in medical and many other fields. To advance the development of functional molecules, research efforts focus on two principal platforms: the originally developed cell-free protein display system “Peptide ligase-mediated display (PL-display)” and an in-house developed injection-molded microwell array chip that enables PL-display-based biochemical activity screening. Based on these platforms, functional screening of protein and peptide variants and derivative technologies are being further developed.

Peptide ligase-mediated display (PL-display)

PL-display is a fully cell‑free protein display technology that covalently links DNA and its encoded protein on magnetic beads through a minimal peptide linker via a peptide ligase reaction. This reaction enables the entirely cell-free construction of protein–DNA linked bead libraries. Because each protein variant is displayed on an individual bead, the library can be observed, evaluated, sorted, and manipulated at the single-variant (single-bead) level. [PNAS Nexus 5, pgag031 (2026)]

Affinity screening at the single-variant level using FACS

By mixing fluorescently labeled target molecules with a PL-display bead library and subjecting the mixture to FACS (Fluorescence-Activated Cell Sorting), beads that have bound the target (i.e., beads displaying proteins with target-binding capability) can be quantitatively detected, sorted, and collected. Optimization of the FACS gating thresholds enabled up to a 10,000-fold enrichment of target-binding genes. [PNAS Nexus 5, pgag031 (2026)]

Biochemical activity screening using injection-molded microwell array chips

A manufacturing process has been established for injection-molded polymer chips containing one million micrometer-scale cubic microwells within a 1 cm² area, produced using an ultra-precision machined mold. By distributing a PL-display bead library into the microwells in a one-bead-per-well loading and measuring biochemical reactions within each well, this platform enables biochemical activity profiling of protein libraries comprising up to one million variants. Sealing microwells in an open-top format and magnetically retrieving beads make it possible to execute directed-evolution cycles.

Keywords

Evolutionary Molecular Engineering, protein display, microwell arary, in vitro selection, FACS-based selection, on-chip selection

Biography

Education

• Mar. 2002

  B.E. in Functional Materials Science from Saitama Univ.

• Mar. 2004

  M.E. in Functional Materials Science from Saitama Univ.

• Sep. 2007

  Ph.D. in Evolutionary Molecular Engineering from Saitama Univ. (Prof. Yuzuru Husimi)

Appointments

• Aug. 2007-Mar. 2010

  Project Researcher, Lab. of Cell Biology, Saitama Univ. (Prof. Takafumi Sakai)

• Apr. 2010-Mar. 2012

  Project Researcher, Biopolymer Engineering Lab., Saitama Univ. (Prof. Naoto Nemoto)

• Apr. 2012-Mar. 2015

  Project Researcher, NanoBioDevice Lab., The Univ. of Tokyo (Prof. Takanori Ichiki)

• Apr. 2015-Mar. 2019

  Part-time Lecturer, Saitama Univ.

• Apr. 2015-Present

  Deputy Principal Research Scientist, Innovation Center of NanoMedicine (iCONM)

Performance

[Original Articles]

1. S. Ueno*, F. Toshioka, T. Ichiki,
   "Peptide ligase-mediated display: A cell-free platform for tunable selection of affinity peptides"
   PNAS Nexus 5, pgag031 (2026) DOI: 10.1093/pnasnexus/pgag031
   Press release EurekAlert Science Japan by JST Phys.org Labmate online
2. B. S. Kim, M. Naito, R. Kamegawa, H. J. Kim, R. Iizuka, T. Funatsu, S. Ueno, T. Ichiki, A. Kishimura, K. Miyata,
   "Photo-reactive oligodeoxynucleotide-embedded nanovesicles (PROsomes) with switchable stability for efficient cellular uptake and gene knockdown"
   Chem. Commun. 56, 9477-9480 (2020). DOI: 10.1039/D0CC01750G
   
3. S. Sato, S. Ueno, T. Ichiki
   "A surface modification process for silica microreactor array chips to seal aqueous solutions"
   Jpn. J. Appl. Phys 59, SIIK01 (2020). DOI: 10.35848/1347-4065/ab7adf
4. S. Ueno*, M. Shioya, T. Ichiki
   "Fluorometric measurement of adenosine 5′-triphosphate using exonuclease V activity"
   J. Photopolymer Sci. Technol 31, 699-704 (2018). DOI: 10.2494/photopolymer.31.699
5. S. Ueno*, T. Ichiki
   "Phototriggered control of enzyme reactions toward high-throughput screening on a microwell array chip"
   J. Photopolymer Sci. Technol 30, 657-660 (2017). DOI: 10.2494/photopolymer.30.657
6. S. Ueno*, T. Hirai, S. Sato, M. Biyani, H. Kuramochi, R. Iizuka, T. Akagi, T. Funatsu, T. Ichiki
   "In situ synthesis and immobilization of enzyme molecules on microreactor array chips"
   J. Photopolymer Sci. Technol 28, 719 -726 (2015). DOI: 10.2494/photopolymer.28.719
7. R. Kobayashi, M. Biyani, S. Ueno, S. R. Kumal, H. Kuramochi, T. Ichiki
   "Temperature-controlled microintaglio printing (TC-μIP) for high-resolution micropatterning of transcribed RNA molecules"
   Biosens. Bioelectron. 67, 115-120 (2015). DOI: 10.1016/j.bios.2014.07.050
8. S. R. Kumal, R. Kobayashi, S. Ueno, T. Ichiki
   "Photo-assisted fabrication of ribosome display microarray"
   J. Photopolymer Sci. Technol. 27, 459-465 (2014). DOI: 10.2494/photopolymer.27.459
9. M. Biyani, Y. Tanaka, S. Sato, S. Ueno, T. Ichiki
   "Evaluation of poly(dimethylsiloxane) microreactors for pattern size miniaturization of microintaglio-printing-based protein microarray"
   Jpn. J. Appl. Phys. 53, 06jl04 (2014). DOI: 10.7567/JJAP.53.06JL04
10. M. Biyani, J. Moriyasu, Y. Tanaka, S. Sato, S. Ueno, T. Ichiki
    "Microintaglio printing of in situ synthesized proteins enables rapid printing of high-density protein microarrays directly from DNA microarrays"
    Appl. Phys. Express 6, 087001 (2013). DOI: 10.7567/APEX.6.087001
11. S. R. Kumal, M. Biyani, S. Ueno, T. Akagi, T. Ichiki
    "Simultaneous synthesis and biotinylation of proteins using puromycin-based labeling technology for fabrication of protein array chip"
    Jpn. J. Appl. Phys. 52, 06GK09 (2013). DOI: 10.7567/JJAP.52.06GK09
12. S. Ueno, S. Kimura, T. Ichiki, N. Nemoto
    "Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method"
    J. Biotechnol. 162, 299-302 (2012). DOI: 10.1016/j.jbiotec.2012.09.003
13. S. Ueno*, A. Ono, R. Kobayashi, Y. Tanaka, S. Sato, M. Biyani, N. Nemoto, T. Ichiki
    "Photoassisted recovery of DNA molecules for on-chip directed evolution"
    J. Photopolymer Sci. Technol. 25, 67-72 (2012). DOI: 10.2494/photopolymer.25.67
    THE BEST PAPER AWARD 2013
14. S. Ueno, S. Yoshida, A. Mondal, K. Nishina, M. Koyama, I. Sakata, K. Miura, Y. Hayashi, N. Nemoto, K. Nishigaki, T. Sakai
    "In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display"
    Proc. Natl. Acad. Sci. USA 109, 11121-11126 (2012). DOI: 10.1073/pnas.1203561109
    Highlighted in SciBX (NPG): SciBx 5(27)
15. N. Nemoto, C. Tsutsui, J. Yamaguchi, S. Ueno, M. Machida, T. Kobayashi, T. Sakai
    "Antagonistic effect of disulfide-rich peptide aptamers selected by cDNA display on interleukin-6-dependent cell proliferation"
    Biochem. Biophys. Res. Commun. 421, 129-133 (2012). DOI: 10.1016/j.bbrc.2012.03.130
16. S. Ueno, H. Arai, M. Suzuki, Y. Husimi
    "An mRNA-protein fusion at N-terminus for evolutionary protein engineering"
    Int. J. Biol. Sci. 3, 365-374 (2007). DOI: 10.7150/ijbs.3.365
17. I. Tabuchi, S. Soramoto, S. Ueno, Y. Husimi
    "Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries"
    BMC Biotechnol. 4, 19 (2004). DOI: 10.1186/1472-6750-4-19

[Proceedings]

1. S. Ueno*, Y. Shirakata, M. Shioya, S. Sato, S. Tsuchiya, T. Ichiki
   "Transpeptidase-mediated in-situ covalent immobilization of cell-free synthesized enzyme for on-chip directed evolution"
   Proceedings of the 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, 870-871 (2019).
2. S. Ueno*, T. Ichiki
   "Spatiotemporal phototriggered control of biochemical reactions for on-chip ultrahigh-throughput screening of enzyme activities"
   Proceedings of the 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1301-1302 (2017).
3. R. Wakai, S. Ueno, T. Ichiki
   "In situ covalent immobilization of protein on a chip by sortase-mediated peptide ligation"
   Proceedings of the 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1299-1300 (2017).
4. 上野真吾*、佐藤秀介、倉持宏美、ジェーンアンキタ、若井涼、白形優衣、赤木貴則、一木隆範
   "人工分子進化を目的としたマイクロアレイシステムの開発"
   The Papers of Joint Technical Meeting on “Chemical Sensor” and “Bio Micro Systems”, IEE Japan, CHS-16-032, BMS-16-054, (2016).
5. A. Jain, S. Sato, S. Ueno, T. Ichiki
   "High-density self-assembled bead microarray technology for high-throughput aptamer screening"
   Proceedings of the 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 900-902 (2015).
6. S. R. Kumal, R. Kobayashi, S. Ueno, T. Ichiki
   "Ribosome display microarray using μ-intaglio printing and photo-crosslinking without removal of stop codons from DNA"
   Proceedings of the 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1817-1819 (2014).
7. S. Ueno*, T. Hirai, A. Tamada, M. Biyani, R. Iizuka, T. Funatsu, T. Ichiki
   "Enzyme-immobilized microwell array for on-chip directed evolution of enzymes"
   Proceedings of the 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1802-1804 (2014).
8. S. Sato, T. Fukuda, T. Hirai, S. Ueno, M. Biyani, T. Akagi, T. Ichiki
   "Fabrication of planar microfluidic device for artificial darwinian selection technology"
   Proceedings of the 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1460-1462 (2013).
9. S. Ueno*, R. Kobayashi, M. Biyani, T. Ichiki
   "Protein-DNA conjugate array chip for on-chip directed evolution"
   Proceedings of the 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1463-1465 (2013).
10. S. Ueno*, A. Ono, R. Kobayashi, Y. Tanaka, S. Sato, M. Biyani, N. Nemoto, T. Ichiki
    "Spot-selective DNA recovery from DNA microarray chips for on-chip directed evolution"
    Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 1300-1302 (2012).
11. S. Soramoto, S. Ueno, I. Tabuchi, Y. Husimi
    "Design of various high quality random libraries for in vitro protein evolution"
    Genome Informatics 13, 527-528 (2002).

[Reviews and books]

1. 上野真吾、一木隆範
   『バイオチップの基礎と応用―原理から最新の研究・開発動向まで―』
   伊藤嘉浩 監修（担当：分担執筆、範囲：無細胞タンパク質合成系を利用したタンパク質マイクロアレイ）
   株式会社 シーエムシー出版（2015年10月15日発刊 ISBN: 978-4-7813-1079-4）
2. 上野 真吾、ビヤニ　マニッシュ、佐藤 秀介、ラジ・クマール スバシニ、倉持 宏実、 赤木 貴則、一木 隆範
   「高集積マイクロアレイ研究開発の現状と動向」
   分析化学 64(6), 421-429 (2015).
3. 根本直人、望月佑樹、上野真吾
   「cDNA displayによる分子デザイン－mRNA display（in vitro virus）からcDNA displayへ－」
   生物物理 53, 250-253 (2013).
4. 上野真吾
   『進化分子工学―高速分子進化によるタンパク質・核酸の開発―』
   伏見譲 監修（担当：分担執筆、範囲：進化分子工学による細胞膜受容体結合ペプチドの創製）
   株式会社 エヌ・ティー・エス (2013年10月発刊 ISBN: 978-4-86469-079-9)
5. 上野真吾、坂田一郎、坂井貴文
   特集 細胞膜受容体「グレリン受容体（GHS-R）とアンタゴニスト」
   生体の科学 64 (5), 408-409 (2013)
6. 上野真吾、坂田一郎、坂井貴文
   「進化分子工学的手法による成長ホルモン分泌促進因子受容体拮抗ペプチドの探索」
   比較内分泌学 38(147), 215-217 (2012)
7. S. Ueno, N. Nemoto
   "cDNA display: rapid stabilization of mRNA display"
   Methods Mol. Biol. 805, 113-135 (2012).
8. 上野真吾
   質疑応答「食欲を抑制する人工ペプチドの概要」
   日本医事新報 No.4616 (2012年10月13日発行)
9. 上野真吾、坂井貴文
   増刊号: ホルモンの病態異常と臨床検査「ホルモンの検査 6)アプタマー解析」
   臨床検査 52(11): 1151-1158 (2008)

[Awards]

1. The Society of Photopolymer Science and Technology, The Photopolymer Science and Technology Award, The Best Paper Award 2013,
   "Photoassisted recovery of DNA molecules for on-chip directed evolution"
   S. Ueno, A. Ono, R. Kobayashi, Y. Tanaka, S. Sato, M. Biyani, N. Nemoto, T. Ichiki
   2013年6月
2. 第14回東京大学生命科学シンポジウム優秀ポスター賞
   「酵素の人工進化を目的とした酵素固定化マイクロアレイ」
   上野真吾、平井辰典、ビヤニマニッシュ、飯塚怜、船津高志、一木隆範
   2014年4月
3. 第13回東京大学生命科学シンポジウムポスター賞
   「タンパク質のハイスループット解析を目的としたcDNAディスプレイマイクロアレイ」
   上野真吾、小林遼、佐藤秀介、小野愛子、ビヤニマニッシュ、一木隆範
   2013年6月

[Patents]

1. Title：Magnetic bead recovery method and magnetic bead recovery device
   Inventors：S. Ueno, S. Tsuchiya
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：WO2024-048660
2. Title：Magnetic bead arrangement method and magnetic bead arrangement device
   Inventors：S. Ueno, S. Tsuchiya
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：WO2024-048648
3. Title：Capillary approach detection method and capillary approach detection device
   Inventors：S. Ueno, S. Tsuchiya
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：WO2024-048645
4. Title：Peptide-nucleic acid complex
   Inventors：S. Ueno, T. Ichiki
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：WO2020-095985
   Patent No.：JP7416715B2, US12421627B2
5. Title：Method for manufacturing substrate having modified layer
   Inventors：S. Sato, T. Ichiki、S. Ueno
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：WO2020-013318
   Patent No.：JP7190273B2
6. Title：Measuring method for enzymatic reaction, screening method and measuring device
   Inventors：T. Ichiki, S. Ueno
   Applicants：Kawasaki Institute of Industrial Promotion
   Publication No.：JP2018-117536
7. Title：Nucleic acid detection method, detection probe, microarray, nucleic acid detection kit, nucleic acid-detection probe-capture probe complex, nucleic acid-immobilized carrier, and fluidic device
   Inventors：T. Ichiki, S. Ueno, T. Ueno, T. Funatsu, K. Suzuki
   Applicants：Nikon Corp, University of Tokyo NUC
   Publication No.：JP2015-073523
8. Title：Method of manufacturing protein array or peptide array, method of identifying functional protein or functional peptide, protein array or peptide array, and functional protein or functional peptide identification kit
   Inventors：T. Ichiki, S. Ueno、H. Osawa
   Applicants：Nikon Corp, University of Tokyo NUC
   Publication No.：WO2015-029714
   Patent No.：JP6521255B2, US10400236B2
9. Title：Nucleic acid linker
   Inventors：T. Ichiki, S. Ueno, M. Biyani, R. Kobayashi, H. Shiono
   Applicants：Nikon Corp, University of Tokyo NUC
   Publication No.： WO2014-142020
   Patent No.：JP6020865B2、US10294472B2
10. Title：Method for producing RNA microarray, and RNA microarray
    Inventors：H. Shiono, N. Nemoto, S. Ueno, T. Ichiki
    Applicants：Nikon Corp, University of Tokyo NUC, Saitama University UNC
    Publication No.： JP2013-192493
11. Title：Protein array production method and protein array
    Inventors：N. Nemoto, S. Ueno, T. Ichiki, H. Shiono
    Applicants：Nikon Corp, University of Tokyo NUC, Saitama University UNC
    Publication No.：JP2013-193988
    Patent No.: JP6015894B2
12. Title：Nucleic acid linker
    Inventors：N. Nemoto,S. Ueno, H. Shiono
    Applicants：Nikon Corp, Saitama University UNC
    Publication No.： WO2013-065827
    Patent No.：JP6057185B2
13. Title：Protein-immobilized solid phase, polynucleotide-immobilized solid phase, and nucleic acid recovery method
    Inventors：S. Ueno, N. Nemoto, T. Ichiki, H. Shiono, H. Osawa
    Applicants：Nikon Corp, University of Tokyo NUC
    Publication No.：WO2013-065782
    Patent No.：JP6048975B2、US9334492B2
14. Title：Linker for evolving protein with enzyme-like activity, and method for screening such protein using the linker
    Inventors：N. Nemoto、S. Ueno, T. Fukushima、S. Kumachi
    Applicants：Saitama University UNC
    Publication No.：JP2013-039060
15. Title：Linker for making mRNA/cDNA-protein conjugant, and method for refining nucleotide-protein conjugant using the same
    Inventors：N. Nemoto, S. Ueno
    Applicants：Saitama University UNC
    Publication No.：JP2012-139197
    Patent No.：JP5858415B2
16. Title：New growth hormone secretion-promoting factor receptor-inhibiting peptide
    Inventors:T. Sakai, S. Ueno, K. Nishigaki, K. Miura
    Applicants：Daiichi Sankyo Co Ltd, Saitama University NUC
    Publication No.：JP2011-182726
    Patent No.：JP5697127B2

Affiliation Society

The Biophysical Society of Japan, The Molecular Biology Society of Japan