Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Abstract

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development, and the development of split complementation systems capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

“…Decreasing light intensities or reducing imaging frequency enabled furthermore to reduce the amplitude of the initial fluorescence drop. This set of experiments suggested that the observed reversible photobleaching was likely due to photoisomerization and/or photoejection of the chromophore, as previously proposed to explain the reversible photobleaching observed when labeling FAST and its variants with low concentrations of HBR derivatives 28,23 . This singular photochemical signature might be used advantageously for selective imaging techniques based on kinetic discrimination 29,30 or for single-molecule localization microscopies 31,32 .…”

“…Imaging of semisynthetic chemogenetic reporters in multicellular organisms is however often hampered by the low cell permeability of synthetic probes. HBR derivatives have been previously shown to allow the labeling of FAST and its derivatives in zebrafish embryos thanks to their very high cell uptake and superior ability to diffuse across multicellular systems 13,22,23 . Preliminary experiments in which we quantified the labeling efficiency in live HeLa cells by flow cytometry showed that full labeling of pFAST was achieved at lower chromophore concentrations than FAST, in agreement with its superior binding affinity ( Fig.…”

“…The first yeast library (called library A in this study) of FAST constructed by error-prone PCR using Genemorph II kit (Agilent) was previously described 23 . The second library (called library B in this study) was constructed by DNA shuffling according to different DNA shuffling library protocols 46,47,48 .…”

“…Steady state UV-Vis absorption spectra were recorded using a Cary 300 UV-Vis spectrometer (Agilent Technologies), equipped with a Versa20 Peltier-based temperature-controlled cuvette chamber (Quantum Northwest) and fluorescence spectra were recorded using a LPS 220 spectrofluorometer (PTI, Monmouth Junction, NJ), equipped with a TLC50TM Legacy/PTI Peltier-based temperature-controlled cuvette chamber (Quantum Northwest). Fluorescence quantum yield measurements were determined as previously described using either FAST:HMBR or quinine sulfate as a reference 13,21,50,23 . Reciprocal dilution with protein solution was used so as to keep the protein concentration constant at 40 μM while diluting the fluorogen solution.…”

Production of the Biocommodities Butanol and Acetone from Methanol with Fluorescent FAST-tagged Proteins using Metabolically Engineered Strains of Eubacterium Limosum

“…As different bacteria and proteins show auto uorescence upon excitation with different wavelength [35,71,72], FAST should be considered as a uorescent reporter protein in a large variety of bacteria as the choice of the uorogen can be adapted to the spectral conditions needed for the study. Recently, Tebo et al [73] reported two engineered FAST versions termed greenFAST and redFAST. While greenFAST binds TF Lime, redFAST only binds TF Coral, which we showed can both be used in E. limosum.…”