De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging
- Long Tran
- Steffen Klein
- David Juergens
- Shajesh Sharma
- Justin Decarreau
- Gyu Rie Lee
- Yujia Wang
- Wei Chen
- Asim K. Bera
- Alex Kang
- Jon Woods
- Emily Joyce
- Dionne K. Vafeados
- Nicole Roullier
- Xinting Li
- Bingxu Liu
- Yang Bo
- Edin Muratspahić
- Tim A. Brown
- Jonathan B. Grimm
- Ronak Patel
- Luke D. Lavis
- Julia Mahamid
- Linna An
- David Baker
2026-07-16
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.