Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group

Abstract

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.

Highly chromophoric fluorescent‐labeled methionyl‐initiator tRNAs applicable in living cells

“…Additionally, expression of Tat protein tagged with AVI peptidewas observed in both HeLa cells and cell‐free extracts to confirm translational activity. [ 41 ] The designed C‐terminal biotinylated fluorescent Tat consisted of the N‐terminal fluorophore 5‐FAM (Ex/Em: 492/512 nm) conjugated to the three different complex forms mentioned above. Fluo imaging of HeLa cell extracts demonstrated that PFM‐tRNAi Met complex showed the highest fluorescent Tat expression (Figure 3C), and the NPFM‐tRNAi Met complex showed the lowest (Figure 3E).…”

“…Biotinylated Cy5-Tat-EGFP was detected by binding to Neu-trAvidin beads, using HeLa cell extracts prepared without washing to preserve endogenous components. As previously reported, [32,41] a plasmid expressing the C-terminal fragment of Tat with AVI-tag and His-tag was prepared(Figure 4C and Figure S6). His-tag facilitated GFP binding and Ni-NTA bead based analysis, as confirmed by fluo microscopy.…”

“…His-tag facilitated GFP binding and Ni-NTA bead based analysis, as confirmed by fluo microscopy. [32,41] AVI-tag labeled beads linked to AVI-tagged, Tatfused EGFP were used as controls and imaging markers, showing high fluo intensity as depicted in Figure S6.…”

“…Additionally, expression of Tat protein tagged with AVI peptidewas observed in both HeLa cells and cell-free extracts to confirm translational activity. [41] The designed C-terminal biotinylated fluorescent Tat consisted of the N-terminal fluorophore 5-FAM (Ex/Em: 492/512 nm) conjugated to the three different complex forms mentioned above.…”

Bioorthogonal Reactions in Bioimaging

“…Bioorthogonal labeling schemes that rely on the incorporation of minimal tag bioorthogonalized building blocks and suitably functionalized probes allow minimal linkage error, and very importantly, these are not limited to proteins [ 37 – 39 ]. Synthetically tailored organic fluorophores offer much smaller size and allow greater flexibility in terms of the site specificity of labeling, often providing improved photophysical properties such as a wider spectral range and greater photostability or brightness compared with, e.g., FPs [ 2 , 33 ]. Furthermore, tailored synthetic probes may also address challenges such as auto- and background fluorescence [ 2 ].…”