We synthesized orange-emitting tetramethylrhodamine (TAMRA)-DBCO and far-red-emitting silicon rhodamine (SiR)-DBCO. TAMRA-DBCO enabled the visualization of newly synthesized proteins and their time-dependent degradation throughout the entire cell. SiR-DBCO was similarly effective, but was mainly distributed to the cytoplasm. The time-dependent decrease of TAMRA-DBCO fluorescence intensity in living cells was suppressed by lysosomal enzyme inhibitors and a proteasome inhibitor, suggesting that newly synthesized proteins are degraded via both pathways. Moreover, imaging of drug-induced senescent cells with TAMRA-DBCO suggested that senescent cells have a lower protein degradation ability than non-senescent cells.
See details in Sumitani et. al. (2026) Bioconjug Chem.
Shun Sumitani, Eita Sasaki, Hisashi Ohno, Sota Yamada, Orie Takayama, Fan-Yan Wei, Yoshihiko Kuchitsu, Tomohiko Taguchi, Kenjiro Hanaoka (2026) Visualizing Newly Synthesized Proteins and Their Degradation Dynamics by Using Long-Wavelength-Emitting Fluorescent Dye-DBCO Conjugates., Bioconjugate chemistry
Published in 2026 Apr 24 (Electronic publication in April 24, 2026, midnight )
(Abstract) Understanding the spatiotemporal dynamics of protein synthesis and degradation is important for establishing how cells maintain protein homeostasis. Conventional methods for detecting newly synthesized proteins include metabolic labeling with radioactive [(35)S]methionine (Met) or the incorporation of l-azidohomoalanine (AHA) or l-homopropargylglycine followed by fluorescent labeling via copper(I)-catalyzed click chemistry. However, these methods typically require cell fixation, making them unsuitable for live-cell imaging. Here, we describe a fluorescence imaging technique to monitor newly synthesized proteins in living cells by utilizing a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, in which l-AHA-containing proteins are labeled with fluorescent dyes conjugated to dibenzocyclooctyne (DBCO). We synthesized orange-emitting tetramethylrhodamine (TAMRA)-DBCO and far-red-emitting silicon rhodamine (SiR)-DBCO. TAMRA-DBCO enabled the visualization of newly synthesized proteins and their time-dependent degradation throughout the entire cell. SiR-DBCO was similarly effective, but was mainly distributed to the cytoplasm. The time-dependent decrease of TAMRA-DBCO fluorescence intensity in living cells was suppressed by lysosomal enzyme inhibitors and a proteasome inhibitor, suggesting that newly synthesized proteins are degraded via both pathways. Moreover, imaging of drug-induced senescent cells with TAMRA-DBCO suggested that senescent cells have a lower protein degradation ability than nonsenescent cells. These methods should be useful for investigating protein homeostasis in living cells.