RNA Structure-Function Properties in Facilitated Hammerhead Ribozymes with Unprecedented Catalytic Rates

Abstract

Purpose : A novel Facilitated hammerhead ribozyme (F-hhRz) (F-hhRz-A7U-266) is a therapeutic ribozyme candidate that attacks full length human rhodopsin mRNA at CUC↓ 266, a position shown to be accessible for RNA annealing and cleavage. RNA structural interactions that form the basis for the orders of magnitude improvement in catalysis are investigated.

Methods : Substrate RHO-266 RNA is 15 or 14 nt long with 5’ FAM and 3’ BHQ1 (IDT); cleavage at CUC↓ 266 results in liberation of quenched fluorescence which is quantified optically. F-hhRz are transcribed (T7pol) from PCR templates or generated synthetically (IDT). Mutations in substrate or/and F-hhRz assess structure-function relationships. Reactions are initiated by mixing F-hhRz with substrate RNA in buffer (10 mM Tris-HCl, pH 7.5) at 0.5 mM Mg²⁺ and optically measured at 37°C in a qRT/PCR machine. Data are analyzed in Origin 8.

Results : Target accessibility, combined with F-hhRz upstream antisense-substrate helix interaction with the 4 nt Stem-II loop cap, promoted enhanced cleavage rates of F-hhRz 266 (143.4 +/-17.3 min^-1) vs hhRz 725 having a structured substrate without hhRz/substrate interactions (11.2 +/- 1.6 min^-1, p=4.46E-7). A F-hhRz 725 with engineered substrate accessibility and hhRz/substrate interactions facilitates cleavage (159.2 +/-12.5 min^-1) similar to F-hhRz 266 (p=0.14). A single base change (A7U) in the 5’ anti-sense flank of F-hhRz 266 significantly increased cleavage rate (329.3 +/-28.8 min^-1, p=1.26E-15). Mutagenic analysis of the A7U F-hhRz Stem-II loop showed strong variation of catalytic activity depending upon nt replacement (p=0). A single U7 nt deletion in the 266 substrate removed a predicted Stem-II loop interaction and strongly suppressed cleavage (90.6 +/-3.2 min^-1, p=9.26E-14), but partial recovery occurred with Stem-II loop variations (p=2.19E-14).

Conclusions : Rate acceleration is due to the hybridized upstream F-hhRz antisense flank and downstream substrate element interacting with the 4 nt loop capping Stem-II of the hhRz. This interaction is hypothesized to align the F-hhRz enzyme core into productive conformational states for efficient nucleic acid based chemistry. F-hhRzs have substantial potential as RNA therapeutics because they can reduce target mRNA levels at lower levels of expression, and function at cellular levels of free Mg²⁺.

This is a 2020 ARVO Annual Meeting abstract.