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

Authors

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

Published in

Science (New York, N.Y.). Pages eaeb0822. Jul 16, 2026. Epub Jul 16, 2026.

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 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.

PMID:
42461986
Bibliographic data and abstract were imported from PubMed on 17 Jul 2026.

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