A far-red fluorescent chemogenetic reporter for in vivo molecular imaging

Abstract

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae and chicken embryo. Beyond enabling genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.

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“…The fluorogen itself penetrates cells extremely fast, which produces quasi-immediate fluorescence upon fluorogen addition. FAST comes with a versatile array of fluorogens and tags, the combination of which forms a comprehensive toolbox suitable for a large array of applications (Plamont et al, 2016;Li et al, 2017;Li et al, 2018;Tebo et al, 2018;Tebo and Gautier, 2019;Li et al, 2020;Benaissa et al, 2021;Tebo et al, 2021). The latest tag variant, pFAST, was engineered from the original tag Y-FAST by directed evolution and displays increased fluorogen binding affinity as well as fluorescence brightness, and is additionally compatible with a collection of fluorogens that span the visible spectrum (Benaissa et al, 2021).…”