Fig. 1: Sequence engineered sup-tRNAArg suppresses RPE65-R44X mutation in HEK293 cells.
a Schematic showing that sup-tRNAArg engineered from near-cognate natural tRNAArg recognizes UGA stop codon by the mutated anticodon. Created in BioRender. Yang, Y. (2025) https://BioRender.com/sb411h1. b Schematic of the workflow used to evaluate the readthrough efficacy of sup-tRNAArg with plasmid constructs encoding the dual-luciferase reporter containing RPE65-R44X sequence context. Created in BioRender. Yang, Y. (2025) https://BioRender.com/sb411h1. c Readthrough efficacy of sup-tRNAArg calculated using the ratio of normalized Gaussia luciferase (Gluc) activity to firefly luciferase (Fluc) activity. Data are mean ± s.e.m. of eight biological replicates. d Nucleotide substitutions in the anticodon-stem (A1-A6 variants) or TΨC-stem (T1-T4 variants). e Comparison of PTC suppression efficiency of TCT-1 and its variants. The readthrough activity was calculated using the ratio of Gluc activity to Fluc activity. Data are mean ± s.e.m. of four biological replicates. f Comparison of PTC suppression efficiency of combined variants and TCT-1. The readthrough activity was calculated using the ratio of Gluc activity to Fluc activity. Data are mean ± s.e.m. of three biological replicates. g Schematic of the workflow used to evaluate the PTC suppression activity of sup-tRNAArg with plasmid constructs encoding the full-length mRPE65-R44X cDNA. Created in BioRender. Yang, Y. (2025) https://BioRender.com/sb411h1. h Representative western blot images (top) and quantification (bottom) of mouse RPE65 protein expression restored by sup-tRNAArg. Data are mean ± s.e.m. of five biological replicates. i qPCR quantification of mRPE65 cDNA in HEK293 cells received indicated treatment. Data are mean ± s.e.m. of five biological replicates. Statistical analysis was performed by two-sided t-test.
