Extended Data Fig. 1: Evaluation of UGA-sup-tRNAArg candidates in HEK293 cells.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
(a) Schematics showing the sup-tRNA construct with a U6 promoter and the dual-luciferase (dual-luc) PTC reporter construct. CLuc: Cypridina luciferase. T2A: Thosea asigna virus 2 A peptide. GLuc: Gaussia luciferase. pA: polyadenylation signal. (b) Bar graph showing the readthrough efficiency as quantified by measuring GLuc activity normalized to CLuc activity in the culture medium. (c) Schematics showing the tandem (2x) sup-tRNA gene constructs with either a U6 promoter (U6-sup-tRNA) or a natural flanking sequence (NFS-sup-tRNA), and the digenic PTC reporter construct with a bi-directional promoter (BiP) driving the expression of mutant Gaussia Luciferase (GLuc) and Enhanced Green Fluorescent Protein with a FLAG tag (EGFP-FLAG), both of which are disrupted with an in-frame TGA nonsense mutation. pA: polyadenylation signal. (d) Workflow of the in vitro readthrough assay with plasmid co-transfection in HEK293 cells. (e) Bar graph showing the readthrough efficiency as quantified by measuring GLuc activity in the culture medium. (f) Bar graph showing the readthrough efficiency as quantified by measuring EGFP fluorescence signal in HEK293 cells. (g) Representative EGFP fluorescence images of HEK293 cells transfected with indicated plasmids. Scale bar: 200 µm. In b, e, f, protein expression from HEK293 cells transfected with a WT reporter plasmid (no TGA nonsense mutations) and an mCherry-only plasmid (no sup-tRNA) was defined as 100%. Each dot represents a biological repeat (n = 3). Data are presented as mean and standard deviation. Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparisons test. Schematic in d created in BioRender. Zou, H. (2025) https://BioRender.com/dqjni51.
