Fluorescence-Based Kinetic Measurements for RNA-Cleaving DNAzymes

Hannah Rosenbach; Gerhard Steger

doi:10.1007/978-1-0716-2047-2_5

Fluorescence-Based Kinetic Measurements for RNA-Cleaving DNAzymes

Methods Mol Biol. 2022:2439:65-77. doi: 10.1007/978-1-0716-2047-2_5.

Authors

Hannah Rosenbach¹, Gerhard Steger²

Affiliations

¹ Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
² Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. steger@biophys.uni-duesseldorf.de.

PMID: 35226315
DOI: 10.1007/978-1-0716-2047-2_5

Abstract

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.

Keywords: Data analysis; Förster resonance energy transfer (FRET); Kinetic measurements; Observed rate constant (kobs); RNA cleavage.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA, Catalytic* / chemistry
Fluorescence
Kinetics
RNA / chemistry
RNA Cleavage

Substances

DNA, Catalytic
RNA