Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.
Bibliographical noteFunding Information:
Funding: This work was supported by the Basic Science Research Program and through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF 2019R1I1A1A01043047).
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.
- bioorthogonal reactions
- carboxyl fluorescent dye
- cell-permeable fluorescent dye
- highly efficient fluorescence labeling
- nickel-nitrilotriacetic acid bead assay
- single native proteins of interest
ASJC Scopus subject areas
- Molecular Biology
- Computer Science Applications
- Physical and Theoretical Chemistry
- Organic Chemistry
- Inorganic Chemistry