Fig. 2

Validation and spectral separation of single fluorescent non-formylated methionine-conjugated initiator tRNA (Met-tRNAi) and production of fluorescent biotin-labeled Tat proteins using non-formyl methionine Escherichia coli initiator tRNA (E. coli Met-tRNAi) and E. coli methionyl tRNA synthetase (EcMRS) in HeLa cells. A Chromatogram spectra for unlabeled E. coli initiator tRNA (tRNAi), L-methionine, and 5-carboxyfluorescein (excitation, 492 nm; emission, 517 nm) resolved using size exclusion high-performance liquid chromatography (SEC-HPLC). Major peaks indicate ultraviolet light alteration (UV; 210 nm). The positive control, E. coli tRNAi, and human tRNAi are shown in black (UV; 260 nm). B The highest peak corresponds to fluorescent Met-tRNAi (UV 210 nm; red line, UV 260 nm; black line, fluorescence detection [FLD]; blue line). The positive control with tRNAi (UV; 260 nm) was used to verify the quality of the fluorescent Met-tRNAi shown in black (UV; 210 nm, FLD; excitation, 492 nm; emission, 517 nm). C Comparison of E. coli tRNAi (green line in 2C) and E. coli fluorescent Met-tRNAi (blue) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. D The activity of the sequence/structure and cellular endogenous human tRNAis activity of the sequence/structure in mammalian cells compared with that in E. coli tRNAis. The orthogonality of replacing endogenous human tRNAi with E. coli tRNAi in association with human methionyl tRNA synthetase (HMRS) and EcMRS-mediated fluorescent biotinylated Tat expression was analyzed using immobilized quartz slides coated with streptavidin. Image of fluorescent biotinylated Tat shows binding of EcMRS to E. coli tRNAi. The E. coli Met-tRNAi/EcMRS pairs and E. coli Met-tRNAi/HMRS pairs were confirmed using fluorescent Tat expression (excitation, 492 nm; fluorescent emission of Tat, 517 nm)