Related Experiment Video
Updated: May 24, 2025

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes
Published on: July 22, 2014
Enhanced hammerhead ribozyme turnover rates: Reevaluating therapeutic space for small catalytic RNAs
Jason M Myers1,2, Jack M Sullivan1,2,3,4,5,6
1Research Service, VA Western NY Healthcare System, Buffalo, NY 14215, USA.
Abstract:
We discovered enhanced hammerhead ribozyme (EhhRz) kinetic performance during therapeutic optimization of a lead agent against the human rhodopsin mRNA target. Turnover activity in trans was greater than 300 nMmin-1 under 10-fold substrate excess and physiological Mg2+ (1 mM). We validated a moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles. The EhhRz targets a CUC↓ cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classical Michaelis-Menten turnover behavior with efficiency (V max/K m ) up to 3.2 × 106 min-1M-1. EhhRzs show cooperative Mg2+ titration (K Mg1/2 = 0.7 ± 0.02 mM; Hill = 1.7 ± 0.1). Structure-function assays showed that the upstream EhhRz antisense flank (substrate bound) interacts with stem-loop II. Tetraloop sequence variation reveals a marked effect on turnover rate. Downstream substrate U7 is not essential for enhanced activity. Single-turnover reaction rates show substantial improvements in hhRz rates at physiological Mg2+. Embedded within a target-reporter fusion mRNA, EhhRzs cleave regional target elements under intracellular conditions promoting strong knockdown at target mRNA and protein levels. EhhRzs have potential as druggable nucleic acid therapeutics against arbitrary targets, or in the design of improved aptazymes.
Related Concept Videos
Ribozymes
Ribozymes can...
Turnover Number and Catalytic Efficiency
Chymotrypsin is a pancreatic enzyme that breaks down proteins during digestion....
Riboswitches
The aptamer has high specificity for a particular metabolite which allows riboswitches to specifically regulate...
Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique...
Improving Translational Accuracy

