Journal Article

Design of Orthogonal Far-Red, Orange and Green Fluorophore-binding Proteins for Multiplex Imaging

Klein, Steffen, Baker, David, Woods, Jon, Liu, Bingxu, Vafeados, Dionne K, An, Linna, Kang, Alex, Lavis, Luke D, Sharma, Shajesh, Roullier, Nicole, Joyce, Emily, Chen, Wei, Bera, Asim K, Tran, Long, Wang, Yujia, Juergens, David, Mahamid, Julia, Decarreau, Justin, Lee, Gyu Rie

2025

Overview

Fluorescent proteins and small molecule dyes have complementary strengths for biological imaging: the former are genetically manipulatable enabling tagging of specific proteins and detection of protein interactions, while the latter have greater photostability and brightness but are difficult to target. To combine these strengths, we used de novo protein design to generate binders to three bright, stable, cell-permeable dyes spanning the visible spectrum: JF657 (far red), JF596 (orange-red) and JF494 (green). For each dye, we obtain nanomolar binders with weak or no binding to the other two dyes; the accuracy of the design approach is confirmed by a crystal structure of one binder which is very close to the design model. Fusion of the JF567, JF596 and JF494 binders to three different targets followed by staining with the three dyes simultaneously enables multiplex imaging. We further expand functionality by incorporating an active site carrying out nucleophilic aromatic substitution to form a covalent linkage with the dye, and developing split versions which reconstitute fluorescence at subcellular locations where both halves are present, enabling both protein-protein interaction detection and chemically induced dimerization with fluorescence reporting. Our designs combine the advantages of fluorescent proteins and small molecule dyes and should be broadly useful for cellular imaging.

• Publisher: Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Preprints
• Subject: Biochemistry