Fig. 1: Screening orthogonal oSD·oASD pairs with two-sided orthogonality in vitro.
A Four types of orthogonalities. SD, Shine–Dalgarno sequence; ASD, anti-Shine–Dalgarno sequence; oSD, orthogonal SD; oASD, orthogonal ASD. B Experimental scheme to screen oSDs that do not interact with native ribosomes in cell extracts. Fluorescence was detected using fluorescence microplate readers. C oSD selection. Either a WT-SD–sfGFP or an oSD–sfGFP reporter (named a, b, c, d, or1, or4, and j) was mixed with S12 or S30 cell extracts. NC, negative control without a reporter. Mean ± SD (n = 3). **, p < 0.01; n.s., not significant; one-way ANOVA with Dunnett’s test against NC. D Further oSD selection. Either a WT-SD–LacZ or an oSD–LacZ reporter (b, or1, or4, and j) was mixed with S12 cell extracts. a.u., arbitrary unit. Mean ± SD (n = 3). One-way ANOVA with Dunnett’s test against NC. E Experimental scheme to screen oSD·oASD pairs with two-sided orthogonality in cell extracts. Cell extracts were prepared using BL21 StarTM (DE3) lacZ::frt expressing an artificial rRNA operon with WT-ASD or oASD (b, or1, or or4) and C1192U spectinomycin resistance (SpcR). F Screening oASDs that do not interact with the WT-SD–LacZ reporter. The cell extracts were mixed with the WT-SD–LacZ reporter and spectinomycin. Mean ± SD (n = 3). *, p < 0.05; two-tailed Welch’s t-test. G Screening oSD·oASD pairs with two-sided orthogonality. The cell extract was mixed with the cognate oSD–LacZ reporter and spectinomycin. Mean ± SD (n = 3). ***, p < 0.001; two-tailed Welch’s t-test. Three biological replicates were used in all experiments. Source data are provided as a Source Data file.
