Stability and Activity of the 10-23 DNAzyme Under Molecular Crowding Conditions

Abstract

DNAzymes are biocatalysts that have been selected in vitro and their function inside cells (in vivo) is extremely low. Thus, almost all studies have been carried out in diluted solutions (in vitro). The cellular presence of molecules such as amino acids, polypeptides, alcohols, and sugars introduces forces that modify the kinetics and thermodynamics of DNAzyme-mediated catalysis. The crowded intracellular environment referred to as molecular crowding can be mimicked by adding high concentrations of natural or synthetic macromolecules to the reaction conditions. Here, we investigate the activity of the 10-23 DNAzyme and the stability of the DNAzyme:RNA complex under molecular crowding conditions. Therefore, we use a Förster resonance energy transfer (FRET)-based activity assay in combination with denaturing urea polyacrylamide gel electrophoresis and circular dichroism (CD) spectroscopy.

Keywords: 10–23 DNAzyme; Circular dichroism (CD) spectroscopy; Denaturation temperature; Deoxyribozymes; Förster resonance energy transfer (FRET); Molecular crowding; Polyethylene glycol (PEG).