De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging
Year: 2026
Type: article
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.
Source: Science
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
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: hybrid
Awards: G1040084506, G1683263358, G2197713987, G2380337622, G3467705078, G3648237380, G3684661152, G6081469444, G6124569277, G6249670178, G6433473157, G697969094, G6980936658, University of Washington Foundation - to support structure based immunogen design for next-generation vaccines, G7951213403, G8118760452, G8217528581, G86151789, G8861629861
OpenAlex ID: W7169058951