De novo design of orthogonal far-red, orange, and green fluorophore-binding proteins for multiplexed imaging
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.
Article Details
Journal Info
Science
American Association for the Advancement of Science
Authors (25)
Long Tran
Steffen Klein
Molecular Systems Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
David Juergens
Shajesh Sharma
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Justin Decarreau
Gyu Rie Lee
Yujia Wang
Wei Chen
Asim K. Bera
Alex Kang
Jon Woods
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Emily Joyce
Dionne K Vafeados
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Nicole Roullier
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Xinting Li
Bingxu Liu
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Yang Bo
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Edin Muratspahić
Tim A. Brown
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Jonathan B. Grimm
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Ronak Patel
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Luke D. Lavis
Julia Mahamid
Molecular Systems Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Linna An
David Baker