De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging
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 further design a two-chain NovoTag 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, expanding the toolkit for cellular imaging.
Authors
- Edin Muratspahić (ORCID: https://orcid.org/0000-0002-6011-0805)
- Asim K. Bera (ORCID: https://orcid.org/0000-0001-9473-2912)
- Yujia Wang (ORCID: https://orcid.org/0000-0002-7099-6283)
- Bo Yang (ORCID: https://orcid.org/0000-0001-5617-5748)
- Julia Mahamid (ORCID: https://orcid.org/0000-0001-6968-041X)
- Bingxu Liu (ORCID: https://orcid.org/0000-0002-3661-3410)
- Dionne Vafeados (ORCID: https://orcid.org/0000-0002-2560-8907)
- Long Tran (ORCID: https://orcid.org/0000-0001-9723-3529)
- Luke D. Lavis (ORCID: https://orcid.org/0000-0002-0789-6343)
- Justin Decarreau (ORCID: https://orcid.org/0000-0001-8996-7741)
- Alex Kang (ORCID: https://orcid.org/0000-0001-5487-0499)
- Timothy A. Brown (ORCID: https://orcid.org/0000-0001-8941-7678)
- Jonathan B. Grimm (ORCID: https://orcid.org/0000-0003-0331-4200)
- David A. Baker (ORCID: https://orcid.org/0000-0001-7896-6217)
- Ronak Patel (ORCID: https://orcid.org/0000-0002-6945-6466)
- Shajesh Sharma (ORCID: https://orcid.org/0000-0001-9829-8631)
- Steffen Klein (ORCID: https://orcid.org/0000-0002-8046-0474)
- Jonathan Woods (ORCID: https://orcid.org/0000-0002-5457-2464)
- Emily Joyce (ORCID: https://orcid.org/0009-0007-3230-1341)
- Xinting Li (ORCID: https://orcid.org/0000-0001-5559-7578)
- David Juergens (ORCID: https://orcid.org/0000-0001-6425-8391)
- Gyu Rie Lee (ORCID: https://orcid.org/0000-0002-9119-5303)
- Wei Chen (ORCID: https://orcid.org/0000-0002-5255-4166)
- Linna An (ORCID: https://orcid.org/0000-0001-9858-9105)
- Nicole Roullier
Institutions
- European Molecular Biology Organization (DE)
- Howard Hughes Medical Institute (US)
- Korea Advanced Institute of Science and Technology (KR)
- Janelia Research Campus (US)
- University of Washington (US)
- European Molecular Biology Laboratory (DE)
- European Molecular Biology Laboratory (DE)
- Stanford University (US)
Publication Details
- Journal
- Science
- Published
- 2026-07-16
- DOI
- https://doi.org/10.1126/science.aeb0822
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00