Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

ABSTRACT We discovered an enhanced functionality hammerhead ribozyme ( EhhRz ), designed to act in trans against human rod opsin ( RHO ) mRNA, with turnover activity >300 nM min −1 under substrate-excess conditions and physiological Mg 2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC↓ cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess ( V max / K m up to 1.60 × 10 8 min −1 M −1 ), which is comparable to RNase A. EhhRzs show cooperative titration with a K d of 0.73 ± 0.02 mM at cellular Mg 2+ concentrations and a Hill coefficient of 1.73 ± 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min −1 . These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic.