Development of bioorthogonal fluorogenic reporters for biological imaging

Abstract : Studying protein activities could help us to understand the complex mechanisms controlling cells and organisms. Our laboratory recently developed Fluorescence-Activating and absorption-Shifting Tag (FAST), a small fluorogen-based reporter enabling to fluorescently label fusion proteins in living cells. My PhD thesis presents the developments of new FAST systems with various properties for multiplexed imaging. We report a collection of fluorogens enabling to tune the fluorescence color of FAST from green-yellow to orange and red. Beyond allowing multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST’s reversible labeling, opening great prospects for the design of selective imaging methods relying on dynamic systems. In order to further expand the spectral properties of FAST to red, we also designed and developed a library of red fluorogenic dyes, for which we engineered specific protein binders by applying a directed evolution strategy based on the yeast display technology and high-throughput fluorescence activating cell sorting (FACS). We finally developed novel fluorogens able to form fluorescent complexes with FAST, but incapable of crossing the plasma membrane, which makes it possible to selectively detect FAST-tagged cell-surface proteins.
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Chenge Li. Development of bioorthogonal fluorogenic reporters for biological imaging. Theoretical and/or physical chemistry. PSL Research University, 2017. English. ⟨NNT : 2017PSLEE044⟩. ⟨tel-01816389⟩

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