Ribozymes designed according to Mouse gut metagenome ribozyme structure. (A) Design template and secondary structure of the “mouse gut metagenome HHRz” structure used as the input for Enzymer. All of the original sequences were modified except the red nucleotides forming the catalytic core. (B) Secondary structure with a new sequence generated by Enzymer, with a color scheme corresponding to that of the design template in A, with mutations compatible or incompatible with the pseudoknot illustrated. Cleavage site is shown by an arrowhead. (C) Cleavage % of the ribozymes during transcription (for 2.5 h) for the self-cleaving ribozymes compared to the WT. (D) Cleavage extent of the cis-acting ribozymes (WT, 1 to 13 and mut). For Rzs 13 mut, 13 mut c, and N/O (non-optimized), the cleavage of trans-acting ribozymes was used. Average of three experiments with standard deviation. Comparison of cleavage extent for cis- and trans-acting ribozymes is available in Supplemental Material (Supplemental Fig. S4). (E) Cleavage rate (kobs) of ribozymes.
