Directed evolution and application of catalytic nucleic acids

Abstract

Nature has provided an abundance of enzymes which catalyze the majority of biological processes. Natural enzymes are all protein and RNA, and there is no natural precedent for a natural DNA enzyme (DNAzyme). However, novel catalytic DNA can be evolved in the laboratory by the process of directed evolution, allowing the discovery of molecules with useful applications which carry the intrinsic stability and robust advantages of DNA. In this thesis, I present the discovery and application of a new peroxidase DNAzyme. In a preliminary study, I systematically developed an efficient asymmetric polymerase chain reaction analytical approach to establish a droplet-based microfluidic evolution selection approach. I developed a Droplet Digital PCR (DD PCR) system to encapsulate, amplify, analyze and detect sense-strand DNA. I established a fluorescence-activated droplet sorting (FADS) platform which will need further development to allow real-time selection of peroxidase DNAzyme. Through an alternative strategy I developed a high-throughput solid support coupled directed evolution method to evolve new peroxidase DNAzymes. I discovered the SBDZ-X DNAzyme with several other sequence variants. Subsequent biophysical and biochemical characterisation of truncated SBDZ-X further revealed that the mini SBDZ-X-3 DNAzyme is a highly active and stable peroxidase DNAzyme. The structure was revealed as being parallel G-quadruplex DNAzyme, and the truncated form has better catalytic properties than all peroxidase DNAzyme variants (PS2.M aptamer, [B7]-3-0 aptamer, and EAD2) previously published to date. Further results of circular dichroism (CD), binding affinity, effects of temperature on enzyme activity, structural stability, and kinetic analysis confirmed that the mini SBDZ-X-3 DNAzyme has significant advantages relative to all peroxidase DNAzyme variants. To demonstrate the biomedical application, the mini SBDZ-X-3 was conjugated with the tail of well-characterised DNA aptamers (SYL3C, AS1411), and a new aptamer-guided DNAzyme mediated ex-vivo proximity-based protein labeling approach was developed. Experimental results of confocal microscopy, western blot analysis and LC-MS/MS showed that the hybrid DNAzyme aptamer catalyzed proximity labeling of biotin-tyramide radicals in the vicinity of the cell surface targeted antigens (EpCAM, nucleolin) within 6-12 minutes in fixed cancer cells. The DNAzyme enabled imaging, and allowed enrichment and identification of proteins associated with the EpCAM and nucleolin cell surface biomarker in fixed cancer cells. Further, aptamer-guided DNAzyme (AS1411-mini SBDZ-X-3/T) based biotinylation of nucleolin followed by LC-MS/MS, gene ontology and STRING analysis revealed nucleolin associated membrane proteins were enriched compared to negative control aptamer alone (AS1411). Thus, this study also provides a nucleic acid tool for proteomic characterization of protein complexes. The directed evolution method demonstrated in this thesis is capable of discovering and guiding the molecular evolution of other efficient peroxidase mimicking biocatalyst (DNAzyme or RNAzyme) from a desired library. Aptamer-guided DNAzyme based spatial proteomics further provides a unique alternative approach to discover and map proteins associated with cell surface biomarkers in healthy and diseased cells and tissues.