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Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

Author

Listed:
  • Jiwoong Kwon

    (Institute for Basic Science (IBS))

  • Jong-Seok Park

    (Ulsan National Institute of Science and Technology (UNIST)
    SK Biopharmaceuticals Co., Ltd.,)

  • Minsu Kang

    (Institute for Basic Science (IBS)
    Korea University)

  • Soobin Choi

    (Hankuk University of Foreign Studies)

  • Jumi Park

    (Ulsan National Institute of Science and Technology (UNIST))

  • Gyeong Tae Kim

    (Ulsan National Institute of Science and Technology (UNIST))

  • Changwook Lee

    (Ulsan National Institute of Science and Technology (UNIST))

  • Sangwon Cha

    (Hankuk University of Foreign Studies)

  • Hyun-Woo Rhee

    (Ulsan National Institute of Science and Technology (UNIST)
    Seoul National University)

  • Sang-Hee Shim

    (Institute for Basic Science (IBS)
    Korea University)

Abstract

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

Suggested Citation

  • Jiwoong Kwon & Jong-Seok Park & Minsu Kang & Soobin Choi & Jumi Park & Gyeong Tae Kim & Changwook Lee & Sangwon Cha & Hyun-Woo Rhee & Sang-Hee Shim, 2020. "Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy," Nature Communications, Nature, vol. 11(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-019-14067-4
    DOI: 10.1038/s41467-019-14067-4
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    Cited by:

    1. Partha Pratim Mondal, 2020. "Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging," PLOS ONE, Public Library of Science, vol. 15(11), pages 1-15, November.

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