Fig. 2: Identification of Tet-responsive hammerhead ribozymes by amplicon-seq.

a Secondary structure of the Tet-hammerhead ribozyme library design. Blue nucleotides indicate the randomized motif. The arrowhead indicates the cleavage site. A/G mutation at the indicated position renders the ribozyme inactive. b Tet riboswitch construct frequency analysis of the original library and c conservation of complexity following transfection in HEK-293 cells. CV, coefficient of variation. d Volcano plots displaying the significance (FDR) of changes in riboswitch abundance as a function of the change in expression (log₂ fold change). e Correlation of expression induction upon Tet for constructs and associated fold changes reported by Beilstein et al.⁴ (X-axis) with fold changes measured in our screen (Y-axis). f Selected hits from the Tet riboswitch library screen, randomized sequence, mean counts per million (CPM), fold change (FC) and false-discovery rate (FDR) at 25 and 50 µM Tet, respectively. The bar plots show the raw counts under untreated, 25 µM and 50 µM Tet-stimulated conditions (three groups from left to right, n = 8 replicates each). g Functional hit validation in HEK-293 cells (n = 3 experiments, mean ± s.d.); Hh-act/inact: constitutively active/inactive ribozyme control. h Maximal fold changes and p-values measured upon stimulation vs. 0 µM Tet for all tested constructs. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired T-Test, two-tailed). Source data are provided as a Source Data file.