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.

The precise regulatory role in protein synthesis by facilitating interactions with mRNA codons for various tRNA modifications is unclear. We previously reported that enhanced green fluorescent protein (GFP) reduced enhanced GFP mRNA expression in human methionine-conjugated initiator tRNA (tRNAi)/tRNA synthetase pairs under methionine-deficient conditions. Here, we investigated the effect of non-formylated methionine-conjugated Escherichia coli tRNAi on the synthesis of the protein initiation of interest (PII) in HeLa cells under intracellular L-methionine levels. We found that E. coli methionine-tRNAi counteracts human methionine-tRNAi, indicating that E. coli methionyl tRNA synthetase can induce enhanced GFP expression due to increased stability of enhanced GFP mRNA. Both complexes could support translation initiation without being employed to introduce methionine residues in the subsequent elongation steps. The results indicated that E. coli methionine-tRNAi could offset human methionine-tRNAi, and E. coli methionine-tRNAi/methionyl tRNA synthetase pairs can drive enhanced GFP mRNA expression. Unlike the human methionine-tRNAi/methionyl tRNA synthetase pairs that were used as a positive control, the non-formylated E. coli methionine-tRNAi/methionyl tRNA synthetase pairs reduced the expression of enhanced GFP mRNA, resulting in reduced HeLa cell survival. Using tRNAs functions causes of heterologous origin, such as from prokaryotes, and modified, to enhance or suppress the synthesis of specific proteins in eukaryotic organisms into the potential may possess a more prominent advantage of E. coli methionine-tRNAi as approaches that can control PII. This study provides new insights on the E. coli methionine- tRNAi/methionyl tRNA synthetase pair induced PII synthesis and the relative viability of cells could pave the way to regulate ecological/biological systems.

In eukaryotic cells and Escherichia coli, protein synthesis is initiated through non-formylated and formylated methionine-conjugated initiator tRNA (Met-tRNAi and fMet-tRNAi, respectively). Studies have shown that protein of interest (POI) reduced EGFP mRNA expression in human Met-tRNAi/human methionyl-tRNA synthetase (HMRS) pairs under methionine-deficient conditions. Intracellular L-Met depletion can result in the inhibition of endogenously non-formylated human Met-tRNAi/HMRS in HeLa cells. We investigated the effect of non-formylated Met-conjugated E. coli initiator tRNA (E. coli Met-tRNAi) on POI synthesis in HeLa cells under intracellular L-Met levels. The expression of GFP—a model protein—in L-methionine-deficient HeLa cells, in which endogenous tRNAi was replaced with two kinds of E. coli tRNAi, E. coli Met-tRNAi/E. coli methionyl-tRNA synthetase (EcMRS) and E. coli Met-tRNAi/HMRS, was analyzed. The results indicated that E. coli Met-tRNAi can offset human Met-tRNAi, and E. coli methionyl tRNA synthetase (EcMRS) can drive the expression of EGFP mRNA. The non-formylated E. coli Met-tRNAi/EcMRS pairs reduced the expression of EGFP mRNA, resulting in reduced HeLa cell survival. Our results provide new insights into the potential use of E. coli Met-tRNAi as novel therapeutics that can control POI synthesis initiation. The E. coli Met-tRNAi/MRS pair has the potential to fine-tune protein synthesis in mammalian cells.