Supplementary Figure 3: Detailed experimental guidelines for the selection of designer ribozymes.

The HHR scaffold expression library containing degenerated nucleotides in the desired loop structure is constructed by site-directed PCR-based mutagenesis using the bacterial expression vector as template. The HHR expression library is transformed into bacteria and functional hybrid ribozymes correlating with high-level eGFP expression are enriched by FACS-mediated sorting. The library subpopulation containing HHR variants with highest self-cleavage performance in bacteria is swapped into the corresponding mammalian expression vector and transformed again into bacteria to isolate clonal HHR variants which are subsequently transfected into mammalian cells for validation. The best-in-class designer ribozymes with optimal functional tertiary inter-loop contacts were identified in mammalian cells by correlating low-level SEAP expression and characterized by sequence analysis.