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Color-Changing Fluorescent Barcode Based on Strand Displacement Reaction Enables Simple Multiplexed Labeling

  • Koki Makino
    Koki Makino
    Department of Bimolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
    More by Koki Makino
  • Etsuo A. Susaki
    Etsuo A. Susaki
    Department of Biochemistry and Systems Biomedicine, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
  • Motomu Endo
    Motomu Endo
    Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
    More by Motomu Endo
  • Hiroyuki Asanuma*
    Hiroyuki Asanuma
    Department of Bimolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
    *[email protected]
  • , and 
  • Hiromu Kashida*
    Hiromu Kashida
    Department of Bimolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
    *[email protected]
Cite this: J. Am. Chem. Soc. 2022, 144, 4, 1572–1579
Publication Date (Web):January 20, 2022
https://doi.org/10.1021/jacs.1c09844
Copyright © 2022 American Chemical Society

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    Abstract

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    Fluorescence imaging techniques have contributed to our understanding of various biological phenomenon; however, fluorescence spectral overlap significantly restricts multiplexing capability. Several strategies have been reported to overcome this limitation by utilizing the superior programmability of DNA technologies and nanostructures, but in practice, it remains challenging to achieve broad adoption of these multiplexed detection methods due to the complexities of these DNA designs. Here we report a color-changing fluorescent barcode (CCFB) approach that enables multiple labeling with simple and small nucleic acid structure design based on sequential toehold-mediated strand displacement reaction. The emission color of CCFB can vary in the predetermined sequence so that multiple targets can be detected simultaneously. The CCFB complex is composed of several oligonucleotides, and its color sequence can be easily expanded further. The CCFB approach is easy and time-saving to operate since the irreversible color-changing reaction occurs by simply adding complementary oligonucleotide. We herein developed 27 different CCFB labels, which required only 14 oligonucleotides. We demonstrated that the CCFB system can be used to label multiple targets by attaching CCFB label to polystyrene beads. Moreover, the CCFB can be used to detect intracellular proteins simultaneously when it is attached to antibodies. We expect that this practical platform will be adopted for comprehensive biomolecular imaging in cells.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c09844.

    • Experimental details for preparation of experimental materials, fluorescence measurements, cell culture, fluorescent imaging, and additional figures and tables (PDF)

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