| Summary: | 碩士 === 國立清華大學 === 化學系 === 102 === To study protein functions and dynamics such as motion and localization in living cells, protein labeling by synthetic fluorescent probes is a powerful technique. We developed novel SNAP-tag labeling fluorescent probes using environment-sensitive fluorophore to achieve large fluorescence enhancement, high cellular permeability, quick reaction rate, and no-wash labeling. In our strategy, we attached environment-sensitive fluorophore, 4-sulfamonyl-7-aminobenzoxadiazole (SBD), to O6-benzylguanine (BG), substrate of SNAP-tag protein, to construct fluorogenic probes. The environment-sensitive fluorophore exhibits very weak fluorescence in polar environment while increases fluorescence dramatically as it is introduced to the protein hydrophobic domain. Our method was successfully applied in living cells to image SNAP-tag expressed membrane, cytosol, and nucleus with no additional washout procedure and also feasible for flow cytometric analysis. We believe this method will be an attractive tool for tracking dynamic protein behaviors and various biological studies. Furthermore, we applied the similar probe design to biotin-avidin system (BAS), a high affinity ligand-protein interaction used in versatile immunoassays and tumor cell diagnoses, by changing the BG moiety to biotin. Our aim is to develop a method for signal turn-on protein recognition. So far, we have successfully created several biotinylated probes to achieve good fluorescence enhancement upon binding to avidin. We expect that this method will contribute to a widespread bioanalytical applications.
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