The Effect of a Stem I Substitution on the Cleavage Rate and Efficiency of a Minimal Hammerhead Ribozyme

Abstract

Ribonucleic acid enzymes, or ribozymes, are RNA that is capable of catalyzing biochemical reactions. Hammerhead ribozymes were one of the earliest known classes of ribozymes, and are one of the best characterized. They are of interest for therapeutic use because they can be artificially engineered to bind to and cleave a specific mRNA target sequence with very few off-target effects. Researchers have discovered that minimal hammerhead ribozymes, those without extended stem and loop structures radiating from the core, are still catalytically active. However, cleavage reactions occur with minimal hammerhead ribozymes only at very high, physiologically irrelevant Mg2+ concentrations. It has been hypothesized that if stem I of a minimal hammerhead ribozyme was replaced by the anticodon stem of E. coli tRNA alanine, the cleavage rate of the ribozyme would be significantly increased. The purpose of this study is to determine whether this prediction is correct, and to compare and analyze the dynamics of a wild type hammerhead ribozyme and a hammerhead ribozyme with this mutation. In this study, the transcription procedure for the RNA strands that comprise the HH8 wild type and HH8 JJ hammerhead ribozymes was optimized. Optimal DNA concentrations were determined, and one of two potential transcription buffers was selected for use in the transcription procedure. Additionally, the ribozymes were transcribed, and preliminary cleavage assays performed on both the wild type and mutant ribozymes suggest qualitatively that the mutant ribozyme is functional.