Extended Data Fig. 3: The CuO-CymR system suppresses UGA-sup-tRNA function.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
(a) Schematics showing the constructs expressing NFS-sup-tRNA with a tetracycline operator (TetO) inserted immediately or 20 bp upstream of the leader sequence (L, 6 bp), and the construct expressing tetracycline repressor (TetR). (b) Representative images of EGFP fluorescence in transfected HEK293 cells (left) and quantification of GLuc activity in culture medium (right) to measure readthrough efficiency following co-transfection of indicated plasmids. When the TetR plasmid was not included (- TetR), a plasmid expressing mCherry was used instead to keep the total amount of DNA in co-transfection the same. Each dot represents a biological repeat (n = 3). Statistical analysis was performed with two-sided t-test. (c) Schematics showing the constructs expressing NFS-suptRNA with a cumate operator (CuO) inserted upstream of the mature sup-tRNA template sequence at indicated position and orientation, and the construct expressing Cym repressor (CymR). (d) Representative images of EGFP fluorescence in transfected HEK293 cells (left) and quantification of GLuc activity in culture medium (right) to measure readthrough efficiency following co-transfection of indicated plasmids. When the CymR plasmid was not included (- CymR), a plasmid expressing mCherry was used instead to keep the total amount of DNA in co-transfection the same. In b and d, each dot represents a biological repeat (n = 3). Data are presented as mean and standard deviation. Statistical analysis was performed by two-sided unpaired t-test.
