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

Jiwoong Kwon, Jong Seok Park, Minsu Kang, Soobin Choi, Jumi Park, Gyeong Tae Kim, Changwook Lee, Sangwon Cha, Hyun Woo Rhee, Sang Hee Shim

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)


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.

Original languageEnglish
Article number273
JournalNature communications
Issue number1
Publication statusPublished - 2020 Dec 1

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)


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