Purchase this article with an account.
Jason Myers, Jack M Sullivan; Discovery of a Hammerhead Ribozyme with Enzyme Kinetics comparable to Protein Enzymes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3406.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
A report in 2015 by O’Rourke et al. (Minimal hammerhead ribozymes with uncompromised catalytic activity, J Mol Biol 427:2340-7) showed that a single trans-Hoogsteen base-pair of a substrate Uridine at position N7 (3’ to the cleavage site) to an A residue in Stem-II loop accelerated catalytic activity of the hammerhead ribozyme (hhRz) at 10 mM Mg2+. We tested this finding in our human rhodopsin (hRHO) mRNA targeting hhRz 266 CUC by disrupting its Watson-Crick base pairing with substrate U7. We evaluated if the enhancer RNA element within our novel Facilitated-hhRz-266 improved cleavage rate in these “U7” agents under substrate excess and physiological Mg2+ (0.5-1.0 mM).
Ribozymes and hRHO RNA’s were transcribed (T7pol) from PCR templates. In vitro hhRz cleavage assay used hRHO 15-mer substrate (10-fold molar excess over hhRz) with 5’FAM and 3’BHQ1 (cleavage yields fluorescence) buffered with 10mM Tris pH7.5 and 0.5 mM Mg2+.
Site-directed mutagenesis (SDM) of the hhRz antisense complement to U7 from an A to U (A7U) increased cleavage rate (61/min to 114/min, mean values, p=2.40E-8). SDM of complement U7 to A7G decreased cleavage rate (61/min to 46/min, mean values, p = 0.009). The increased activity of A7U hhRz 266 was likely based on trans-Hoogsteen interactions with hhRz Stem II Loop (GAAA) as alternative loop sequences substantially suppressed cleavage activity: UUCG (mean 27/min, p=1.21E-9) or CUUG (mean 12/min, p=9.63E-10). Catalytic activity of the control hhRz 266 (61/min) enhanced with 3’ addition of a Facilitator RNA element to 91/min (mean value, p=6.17E-8). Catalytic activity of hhRz 266 A7U (114/min) enhanced by Facilitator to 228/min (mean values, p=2.30E-6). A non-cleavable CUG substrate was not cleaved by any hhRz (control). hhRz 266 A7U-Facilitator, under classical reaction conditions (10mM Mg2+), cleaved in excess of 1000/min.
Enzymes evolved to catalyze biological processes by lowering the activation energy to reach a transition state. Several decades of research failed to improve minimal hhRz catalysis in trans under physiological Mg2+ levels and Michaelis-Menten conditions ([S] > [E]) with rates capped at about 1-2/min. Our new hhRz 266 A7U-Facilitator brings the minimal hhRz into a new era of RNA catalysis in trans. These novel properties of Facilitated hhRzs have substantial therapeutic potential for retinal degenerative diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
This PDF is available to Subscribers Only