Abstract
trans‐Cleaving hammerhead ribozyme variants were generated with mimicked non‐conserved internal loop motifs derived from five structurally diverse natural cis‐cleaving ribozymes. Most modified trans‐cleaving variants showed enhanced single‐turnover cleavage rates relative to minimal counterparts that lack tertiary interactions between internal loop motifs I and II, and relative to controls with sequence changes in loop I. The trans‐cleaving ribozyme derived from the positive strand of peach latent mosaic viroid had the highest observed cleavage rate, suggesting a structurally optimized motif that facilitates rapid formation of the ribozyme catalytic center in a trans‐reaction.