Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

Sangyoon Ko, Jiwoong Kwon, Sang Hee Shim

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)


We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

Original languageEnglish
Article number647590
JournalFrontiers in Molecular Biosciences
Publication statusPublished - 2021 Apr 20

Bibliographical note

Funding Information:
We thank Jong-Seok Park in the Ulsan National Institute of Science and Technology for generating the eUnaG-containing plasmids and his advisor, Prof. Hyun-Woo Rhee, in the Seoul National University for kindly providing the genetic constructs. Funding. This work was supported by the Institute for Basic Science (IBS-R023-D1).

Funding Information:
This work was supported by the Institute for Basic Science (IBS-R023-D1).

Publisher Copyright:
© Copyright © 2021 Ko, Kwon and Shim.


  • UnaG
  • bilirubin
  • fluorescent protein
  • photoswitching
  • single-molecule localization microscopy
  • super-resolution (SR) imaging

ASJC Scopus subject areas

  • Biochemistry
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Molecular Biology


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