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Peer-reviewedCC BYScience

De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging

  • Long Tran1,2
  • Steffen Klein3
  • David Juergens4,5,6,2
  • Shajesh Sharma7,2
  • Justin A. Decarreau2
  • Gyu Rie Lee4,8,2
  • Yujia Wang4,2
  • Wei Chen4,2
  • Asim K. Bera2
  • Alex Kang2
  • Jonathan Woods2
  • Emily Joyce2
13 more authors
  • Dionne K. Vafeados2
  • Nicole Roullier2
  • Xinting Li2
  • Bingxu Liu4,2
  • Bo Yang4,2
  • Edin Muratspahić4,2
  • Timothy A. Brown9
  • Jonathan B. Grimm9
  • Ronak H. Patel9
  • Luke D. Lavis9
  • Julia Mahamid10,3
  • Linna An4,2
  • David A. Baker4,11,2
11 affiliations
  1. 1Department of Chemical Engineering, University of Washington, Seattle, WA, USA
  2. 2Institute for Protein Design, University of Washington, Seattle, WA, USA
  3. 3Molecular Systems Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
  4. 4Department of Biochemistry, University of Washington, Seattle, WA, USA
  5. 5Department of Chemistry, Stanford University, Stanford, CA, USA
  6. 6Graduate Program in Molecular Engineering, University of Washington, Seattle, WA, USA
  7. 7Department of Bioengineering, University of Washington, Seattle, WA, USA
  8. 8Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea
  9. 9Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
  10. 10Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
  11. 11Howard Hughes Medical Institute, University of Washington, Seattle, WA, USA
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Abstract

Fluorescent proteins and small-molecule dyes offer complementary advantages for biological imaging: Proteins are amenable to genetic tagging, whereas dyes provide superior brightness and photostability. To combine these strengths, we used de novo protein design to generate small, nanomolar-affinity, high-selectivity binders (NovoTags) for three cell-permeable dyes spanning the visible spectrum. We show that the NovoTag fluorescent lifetimes can be tuned and demonstrate their application in lifetime- and wavelength-based multiplexed fluorescence imaging. We also designed a two-chain version (NovoSplit) that functions as a chemically induced dimerization system with fluorescent readout in living cells or as a minimally perturbing proximity probe in fixed cells. Our approach combines the advantages of fluorescent proteins and small-molecule dyes, thus expanding the toolkit for cellular imaging.

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Cite

BibTeXRIS
BibTeX
@article{tran2026novo,
  title = {{De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging}},
  author = {Long Tran and Steffen Klein and David Juergens and Shajesh Sharma and Justin A. Decarreau and Gyu Rie Lee and Yujia Wang and Wei Chen and Asim K. Bera and Alex Kang and Jonathan Woods and Emily Joyce and Dionne K. Vafeados and Nicole Roullier and Xinting Li and Bingxu Liu and Bo Yang and Edin Muratspahić and Timothy A. Brown and Jonathan B. Grimm and Ronak H. Patel and Luke D. Lavis and Julia Mahamid and Linna An and David A. Baker},
  journal = {Science},
  year = {2026},
  month = jul,
  doi = {10.1126/science.aeb0822},
  url = {https://doi.org/10.1126/science.aeb0822},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging
AU  - Long Tran
AU  - Steffen Klein
AU  - David Juergens
AU  - Shajesh Sharma
AU  - Justin A. Decarreau
AU  - Gyu Rie Lee
AU  - Yujia Wang
AU  - Wei Chen
AU  - Asim K. Bera
AU  - Alex Kang
AU  - Jonathan Woods
AU  - Emily Joyce
AU  - Dionne K. Vafeados
AU  - Nicole Roullier
AU  - Xinting Li
AU  - Bingxu Liu
AU  - Bo Yang
AU  - Edin Muratspahić
AU  - Timothy A. Brown
AU  - Jonathan B. Grimm
AU  - Ronak H. Patel
AU  - Luke D. Lavis
AU  - Julia Mahamid
AU  - Linna An
AU  - David A. Baker
PY  - 2026
DA  - 2026/07/16
JO  - Science
DO  - 10.1126/science.aeb0822
UR  - https://doi.org/10.1126/science.aeb0822
AB  - Fluorescent proteins and small-molecule dyes offer complementary advantages for biological imaging: Proteins are amenable to genetic tagging, whereas dyes provide superior brightness and photostability. To combine these strengths, we used de novo protein design to generate small, nanomolar-affinity, high-selectivity binders (NovoTags) for three cell-permeable dyes spanning the visible spectrum. We show that the NovoTag fluorescent lifetimes can be tuned and demonstrate their application in lifetime- and wavelength-based multiplexed fluorescence imaging. We also designed a two-chain version (NovoSplit) that functions as a chemically induced dimerization system with fluorescent readout in living cells or as a minimally perturbing proximity probe in fixed cells. Our approach combines the advantages of fluorescent proteins and small-molecule dyes, thus expanding the toolkit for cellular imaging.
SN  - 0036-8075
SN  - 0193-4511
SN  - 1095-9203
ER  -