Directed evolution of peroxidase DNAzymes by a function-based approach.

Peroxidase DNAzymes are single-stranded, stable G-quadruplexes structures that exhibit catalytic activity with cofactor hemin. This class of DNAzymes offers several advantages over traditional protein and RNA catalysts, including thermal stability, resistance to hydrolysis, and easy of synthesis in the laboratory. However, their use in medicine, biology, and chemistry is limited due to their low catalytic rates. Selecting and evolving for higher catalytic rates has been challenging due to limitations in selection methodology which generally use affinity as the selection pressure instead of kinetics. We previously evolved a new peroxidase DNAzyme (mSBDZ-X-3) through a directed evolution method, which was subsequently used for proximity labelling in a proteomic experiment in cell culture. Herein, we present a detailed protocol for this function-based laboratory evolution method to evolve peroxidase DNAzymes for future laboratory implementation. This approach is based on capturing self-biotinylated DNA, which is catalyzed by intrinsic peroxidase activity to select for DNAzyme molecules. The selection method uses fluorescence-based real-time monitoring of the DNA pools, allowing for the enrichment of catalytic activity and capture of catalytic DNA across evolutionary selection rounds. The evolved mSBDZ-X-3 DNAzyme attributes parallel G-quadruplex structure and demonstrates better catalytic properties than DNAzyme variants evolved previously. The influence of critical reaction parameters is outlined. This protocol enables discovery of improved peroxidase DNAzyme/RNAzyme variants from natural or chemical-modified nucleotide libraries. The approach could be applicable for the selection of catalytic activities in a variety of directed molecular evolution contexts.