Yarrowia lipolytica ribozymes overlapping RFP coding sequence. (A) Design template and secondary structure of the “Yarrowia lipolytica HHRz” structure used as the input for Enzymer. The original sequence of the ribozyme was modified, except the red nucleotides forming the catalytic core. Nucleotides corresponding to sequence constraints for fusion with coding sequence and Shine–Dalgarno are indicated. (B) Secondary structure with a new sequence generated by Enzymer, with a color scheme corresponding to that of the design template in A. Portions corresponding to sequence of the RFP gene are underlined in yellow for the coding sequence and in orange for the Shine–Dalgarno (SD). Cleavage site is shown by an arrowhead. (C) Cleavage activity of the ribozymes during transcription (for 2.5 h) compared to the WT. Cleaved products of YL1 and YL2 are different from wild type because we reduced stem III by four base pairs and one nucleotide from the loop as compared to WT to permit proper spacing between SD and start codon. (D) The RFP repression assay in which the red fluorescence intensity is measured after 3 h induction with IPTG and compared between the active ribozymes (RzYL 1) and the mutated inactive version (RzYL 1 m), with one control of the unmodified RFP expression (RFP). The bands on the gel, appearing in the uncleaved control lanes (mut), are likely due to a nonspecific degradation of RNA.
