Fig. 4
From: Massively parallel RNA device engineering in mammalian cells with RNA-Seq
RNA-Seq and FACS-Seq of xanthine, folinic acid and c-di-GMP switch libraries. a–c Secondary structures for ribozyme switch libraries designed with the a xanthine aptamer (purple), b folinic acid aptamer (green), and c the binding domain of the cyclic di-GMP-II riboswitch (red). All binding domains are grafted onto stem I of the sTRSV hammerhead ribozyme (gray), while stem loop II consists of 5–6 degenerate bases (yellow). d, f Normalized RNA read count from RNA-Seq assays performed on xanthine, folinic acid, and cyclic di-GMP-II ribozyme switch libraries in the absence and presence of ligand. −/+ Ligand conditions are d 0/5 mM hypoxanthine, e 0/6 mM (6R, S)-folinic acid, f 0/1 mM cyclic di-GMP. Pluses are spiked-in control ribozymes; circles indicate sequences selected for validation. g–i Relative fluorescence values from FACS-Seq assays performed on xanthine, folinic acid, and cyclic di-GMP-II ribozyme switch libraries in the absence and presence of ligand. −/+ Ligand conditions are g 0/5 mM hypoxanthine, h 0/6 mM (6R, S)-folinic acid, i 0/1 mM cyclic di-GMP. Inset Venn diagrams show the number of sequences shared between RNA-Seq and FACS-Seq for sequences with >90th percentile activation ratio. j–l Relative fluorescence values from flow cytometry analysis of individual sequences of the xanthine (j), folinic acid (k), and cyclic di-GMP-II (l) ribozyme switch libraries. Activation ratio of mCherry/BFP (xanthine and cyclic di-GMP) or eGFP/mCherry (folinic acid) at the higher ligand concentration is indicated above each set of bars. sTRSV is the wild-type hammerhead ribozyme; sTRSVctl is a non-cleaving mutant of sTRSV; error bars indicate standard deviation of two or more biological replicates; asterisks indicate the Benjamini–Hochberg corrected p-values of switching significance between 0 mM and the higher ligand concentration using p-values from unpaired, one-tailed t-tests, *p < 0.01, **p < 0.001, ***p < 0.0001, ****p < 0.00001. Filled circles are individual replicate data points. Source data are available in the Source Data file
