Enhanced Hammerhead Ribozymes as Synthetic Nucleic Acid Ocular Therapeutics

Abstract

Purpose : We are investigating structure/function potential with enhanced hammerhead ribozymes (EhhRzs), discovered in this lab, that exceed the turnover rate of historical minimal hhRzs (mhhRzs) (1/min) by at least two log-orders. Therapeutic potential is enhanced because less EhhRz is needed to affect strong levels of target mRNA cleavage vs. a mhhRz. Gene and/or synthetic nucleic acid therapeutics become feasible. Synthetic RNA therapeutics require stabilization against nucleases for intraocular injection.

Methods : Substrate RHO-266 RNA is 15 nt long with 5’ FAM and 3’ BHQ1 (IDT); cleavage at CUC 266 results in liberation of quenched fluorescence which is quantified optically in real time. EhhRzs are transcribed (MegaShortScript) from PCR templates or generated synthetically (IDT). RNA Thermostability Enhancer Set (TriLink) was utilized to evaluate modified nucleotides. Anti-Reverse Cap Analog (ARCA) and poly(A) tailed transcripts were transcribed with mMESSAGE mMACHINE T7 Ultra (ThermoFisher). Reactions are initiated by mixing EhhRz 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 : Site-specific chemical modifications (2’-O-Methyl) of the antisense flanks and the first three residues of the stem-II loop allow enhanced turnover activity (Model 1: 712/min vs unmodified 282/min p=9.4E-17, Model 2: 882/min vs unmodified 746/min p=7.1E-5), whereas modifications in the enzyme core are not tolerated (ANOVA F=386.9, p≈0). ARCA and addition of poly(A) tail to a model EhhRz supports enhanced turnover activity (ANOVA F=363.2, p=2.07E-11). Replacement of all A residues (2-aminoadenosine-5’ppp) obviates activity, whereas replacement of all U residues (5-methyuridine-5’ppp) or replacement of all C residues (5-methycytidine-5’ppp) permits strong enhanced turnover rate of the model EhhRz (U=389/min, C=206/min).

Conclusions : Efforts to stabilize EhhRz RNA with established synthetic chemistries by modifications of the antisense flanks and parts of the stabilizing stem-II loop region, or 5’ capping and 3’ polyadenylation has shown preservation of strong turnover rate activity, under substrate excess conditions and physiological Mg²⁺ levels. Certain enzyme core modifications are tolerable, but other residues critically involved in the reaction mechanism will require alternative chemistries from those already evaluated.

This is a 2021 ARVO Annual Meeting abstract.