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 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.