Fig. 5: Modifications confer enhanced decoding efficiency and reduced immunogenicity to natural tRNA.

a Top, workflow of assessing the decoding ability of tRNAs with 5 × CUG-mCherry mRNA in HEK293T cells. Middle and bottom, schematic diagram illustrating how this method works. Briefly, in normal situation, the successive CUG codons impede the movement of ribosomes due to the lack of tRNALeuCAG. Overexpression of cognate tRNAs alleviates the ribosomal traffic jam in this region and contributes to more rapid translation, the extent of which is associated with the decoding ability of input tRNAs. Figure was generated using BioRender. b, c Representative fluorescent images and quantification of the expression of mCherry using flow cytometry, normalized to that of Lt-CAG. Lst-UUA and Lst-CUA served as the negative controls (n = 3 biological replicates). Scale bars, 100 μm. d Aminoacylation efficacy of tRNAs determined by the in vitro aminoacylation assay (n = 3 biological replicates). e, f HEK293T-TLR8-NF-κB-EGFP cell line was used to measure the immunotoxicity of IVS-tRNA, as indicated from the percentage of EGFP positive cells (n = 3 biological replicates). Left, 0.2 μg of IVS-tRNA; right, 0.4 μg of IVS-tRNA. Figure was generated using BioRender. g TLR-dependent activation by IVS-tRNA was monitored in HEK293T-TLR8 cells with IL-8 secretion as the quantitative indicators (n = 3 biological replicates). Left, 0.2 μg of IVS-tRNA; right, 0.4 μg of IVS-tRNA. h Schematic diagram showing that tRNA immunogenicity was evaluated in M0 macrophages derived from THP-1 cells pretreated with PMA. R848, an agonist that activates both TLR7 and TLR8, served as a positive control. Mock, mock-transfected cells (n = 3 biological replicates). Figure was generated using BioRender. Statistical significance was analysed using one-way ANOVA with Dunnett’s correlation (b, d, f, h). NS, not significant. Data are presented as mean ± s.d. Source data are provided as a Source Data file.