Abstract
Purpose :
Investigate RNA structure/function properties of Enhanced hammerhead ribozymes (EhhRz) to ascertain underlying molecular biophysical properties of log-order kinetic improvements.
Methods :
EhhRz-266 attacks full length human rhodopsin mRNA at CUC↓266, a rare position accessible for annealing and cleavage. Minimal substrate RHO-266 RNA is 15 or 14 nt long with 5’ FAM and 3’ BHQ1 (IDT); cleavage at CUC↓ 266 and product release results in liberation of quenched fluorescence which is quantified optically in real time in a qRT/PCR machine. EhhRzs are transcribed (T7pol) from PCR templates or synthetized (IDT). Mutations in substrate or/and EhhRz assess structure-function relationships in reactions initiated by mixing EhhRz with substrate RNA (100 nM: 1 uM) in buffer (10 mM Tris-HCl, pH 7.5) at 0.5 mM Mg2+ and optically measured at 37°C. Data are analyzed in Origin.
Results :
Variation of Stem-II tetraloops (n=24) had marked impact on kinetic turnover activity relative to wild type (GAAA) with both 15-mer (ANOVA, p = 0) and 14-mer (ANOVA, p=0) substrates. Optimal tetraloops are from a class with unique 3D structure. Changes in the 5’ end of the EhhRz, base pairing with the 3’ end of the substrate, have marked impact on turnover rates at the A7 position (ANOVA, p=0), severe impact at the G6 position (ANOVA, p=0), and less marked impact at the A5 position (ANOVA, p=4.44E-16). Length and composition of Stem-II has a marked impact on turnover rate (ANOVA, p=1.11E-16). The number of G residues at the 5’ end of the EhhRz has a marked impact on turnover rate (ANOVA, p=0). Addition of a structured stem at the 5’ end of the EhhRz is inhibitory (ANOVA, p=0). Variation of the NUH↓ cleavage site impacts turnover (ANOVA, p=0).
Conclusions :
Kinetic efficiency (Vmax/Km) of EhhRzs is on scale of protein RNaseA. EhhRz upstream antisense flank (bound to substrate mRNA) interacts with Stem-II tetraloop to promote catalysis. The optimum Stem-II length (4bp) infers critical distance-dependent interactions. Variation in rate with upstream composition suggests specific nucleotide interactions with Stem-II-tetraloop. Variation in rate with tetraloop infers that these interactions can be highly varied (topological) and dynamic. Outcomes are relevant to establishment of design rules for EhhRzs as nucleic acid therapeutics for arbitrary target mRNAs.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.