A Facilitated Hammerhead Ribozyme Rhodopsin Therapeutic with Catalytic Efficiency on Par with Ribonuclease A at Cellular Free Mg²⁺ Levels

Abstract

Purpose : To rigorously characterize the catalytic efficiency of a novel Facilitated hammerhead ribozyme (F-hhRz) and evaluate its sensitivity to free Mg²⁺ levels. The F-hhRz (F-hhRz-266-A7U-12bp) is a lead therapeutic ribozyme candidate that attacks human rhodopsin mRNA at CUC↓266, a position in the full length mRNA that is accessible to therapeutic RNA annealing and cleavage. F-hhRzs are catalytic RNAs with orders of magnitude improvement in catalysis compared to historical agents.

Methods : Substrate RHO-266 RNA is a 15 nt long substrate with 5’ FAM and 3’ BHQ1 (IDT); cleavage at centered CUC↓ 266 results in liberation of quenched fluorescence which is quantified optically. Ribozyme RNAs with natural nucleotides are also generated synthetically (IDT). Reactions are initiated by mixing F-hhRz with substrate RNA in buffer (10 mM Tris-HCl, pH 7.5) at specified free Mg²⁺ and optically measured at 37°C in a qRT/PCR machine (Cepheid). Data are analyzed in Origin 8 (Microcal).

Results : At 1 μM Substrate the cleavage rate fit with respect to F-hhRz concentration (0-500 nM) shows an expected linear relationship with a slope of 2.18 E9 min^-1M^-1 (R² = 0.991). At 50 nM F-hhRz and 0.5 mM Mg²⁺ the relationship of observed rate (k_obs) with Substrate concentration (0-5000 nM) was well fit by the nonlinear Michaelis-Menten equation (R² = 0.993); fitting of the data to the Eadie-Hofstee linear transformation of the equation (R² = 0.9517) identified V_max = 164 ± 0.25 min^-1 and K_m = 629.9 ± 2.32 nM, leading to an enzyme efficiency (V_max/K_m) = 2.60 E8 min^-1M^-1. At 100 nM F-hhRz and 1 μM Substrate ([S]:[E] = 10:1) the Mg²⁺ sensitivity (0-20 mM) of k_obs is best fit by an independent two-site Boltzmann titration model (R² = 0.995) with approximately equal weights (0.491 ± 0.113) with K_m1 = 0.723 ± 0.058 mM and K_m2 = 5.00 ± 1.12 mM and slope factors k1 = 0.304± 0.054 and k2= 1.648± 0.922; a cooperative two-site Hill model did not fit the data well.

Conclusions : The reported enzyme efficiency of proteinaceous RNaseA enzyme is 1.38 E8 min^-1M^-1. The F-hhRz RNA has a catalytic efficiency that rivals a highly efficient protein enzyme that is thought to use the same reaction mechanism in RNA cleavage. F-hhRzs have substantial potential as RNA therapeutics because they can reduce target mRNA levels at lower levels of their own expression, and they function at cellular levels of free Mg²⁺.

This is a 2020 ARVO Annual Meeting abstract.