RT Journal Article SR Electronic T1 An engineered multifunctional protein tag for advanced fluorescence imaging JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.01.29.428635 DO 10.1101/2021.01.29.428635 A1 Hela Benaissa A1 Karim Ounoughi A1 Isabelle Aujard A1 Evelyne Fischer A1 Rosette Goïame A1 Julie Nguyen A1 Alison G. Tebo A1 Chenge Li A1 Thomas Le Saux A1 Lydia Danglot A1 Nicolas Pietrancosta A1 Xavier Morin A1 Ludovic Jullien A1 Arnaud Gautier YR 2021 UL http://biorxiv.org/content/early/2021/01/31/2021.01.29.428635.abstract AB Fluorescent reporters are essential tools in cell biology for imaging the dynamics of proteins in living cells and organisms with high spatial and temporal resolution. Chemogenetic systems made of a genetically encoded protein tag acting as an anchor for synthetic fluorophores combine the targeting selectivity of genetic tags with the advantages of synthetic fluorophores. Here, we present the directed evolution of a small 14-kDa protein tag with extended chromophore promiscuity capable of efficiently forming non-covalent fluorescent assemblies with a collection of membrane-permeant and membrane-impermeant fluorogenic chromophores displaying spectral properties spanning the entire visible spectrum. The ability to adapt the fluorescence color by choosing a different live-cell compatible fluorogenic chromophore enables to genetically encode blue, cyan, green, yellow, orange and red fluorescence with a single tag, providing an unprecedent experimental versatility. The possibility to form dark assemblies using non-fluorescent chromophores provides moreover an innovative way for switching off fluorescence on-demand in cells and organisms with high temporal resolution by chromophore replacement. Selective wash-free fluorogenic labeling of fusion proteins could be achieved with high efficiency in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms, allowing high contrast imaging with various advanced microscopy techniques. The remarkable labeling efficiency and fluorescence performance allowed the multicolor imaging of dynamic processes in multicellular systems. The ability to match the spectral properties to the imaging modalities and the high photostability enabled to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.Competing Interest StatementThe authors declare the following competing financial interest: A.G. and L.J. are cofounders and hold equity in Twinkle Bioscience/The Twinkle Factory, a company commercializing the FAST technology.