Fluorescence-Based Kinetic Measurements for RNA-Cleaving DNAzymes

Studying the catalytic behavior of biocatalysts under different conditions including temperature, buffer conditions, and cofactor concentrations is an important tool to understand their reaction mechanism. We describe two protocols that allow for the investigation of the catalysis of RNA-cleaving DNAzymes. The techniques include the use of FRET-labeled RNA substrates for studying the RNA-cleavage reaction in real-time under high throughput as well as RNA substrates labeled with a fluorescein molecule at the 5' end for gel-based assays. Both methods allow for an accurate determination of rate constants given a reaction model.