Abstract
Purpose :
We previously discovered that hammerhead ribozymes can be engineered with enhanced kinetic turnover rates (EhhRzs) against short unstructured target RNAs. Here we evaluate the capacity of EhhRzs to cleave structured mRNA targets both in cis, in trans and within the intracellular environment. This is a critical next step in use of catalytic RNAs as clinically-viable post-transcriptional silencing agents.
Methods :
A structured RNA target (51 nt) derived from human rhodopsin mRNA (hRHO) contains a hhRz cleavage site (CUC) at position 266. Using a fusion RNA reporter we embedded four copies (1 in 5’UTR, 3 in 3’UTR) of this target RNA element into an mCherry mRNA expression construct. We subsequently embedded 1, 2, or 4 copies of an EhhRz targeting the 266 cleavage site downstream of the 3’UTR target elements. We also generated a target (1-532 nt) of hRHO for in vitro transcription that contains the native 266 CUC site. In vitro T7pol co-synthesis/cleavage reactions were analyzed by PAGE urea gels. In cellulo functional assays measured suppression of the mCherry fluorescence by quantitative imaging (Keyence). Catalytically inactivated EhhRzs and hardened mRNA targets prevent cleavage. Statistical analysis performed in Origin.
Results :
EhhRzs cleave embedded structured hRHO target elements within the folded mCherry mRNA (>1200 nt) to liberate combinatorial products during in vitro transcription. The extent of cleavage depends upon the number of EhhRzs embedded within the 3’UTR. Catalytic core EhhRz mutations eliminated product bands. Co-synthesis of the mCherry-EhhRz with the independent 532 nt hRHO leads to a robust 266 nt product indicating that the EhhRzs self-cleaving from the in cis target are able to encounter and cleave a structured hRHO target element in trans. Extent of cleavage of the trans target was proportional to the number of EhhRzs embedded and hardening of this target prevents cleavage. Transfection of plasmids expressing the mCherry-EhhRz(4X, active) suppressed mCherry fluorescence by nearly 80% relative to catalytic core mutant controls in HEK293S cells (p<<0.05).
Conclusions :
EhhRzs cleave at accessible regions of structured mRNA targets in cis and in trans and strongly suppress target protein expression by cleavage in untranslated regions that affect mRNA half-life. These are critical next steps for design and development of post-transcriptional EhhRz knockdown therapeutics for ocular diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.