The development of signal amplification detection technology utilising DNAzymes

The principal aims of the research in this thesis were to: (i) Create novel molecular switches using nucleic acid-modifying DNA enzymes (DNAzymes) and (ii) Utilise these to construct an isothermal, signal amplification cascade for the rapid and sensitive detection of target analytes. Such technology is highly desirable for Point of Care (POC) testing, where immediate diagnostic information is required but resources are limited. Two types of molecular switches are described; both achieve temporary inactivation of DNAzymes via hybridisation with blocking oligonucleotides (BL), then each use a unique method to re-activate the DNAzyme. The first switch involves the use of a releaser oligonucleotide to separate and release the DNAzyme from the BL. This strategy was then used as a basis for cascades involving primers that direct the activation and/or synthesis of DNAzymes via strand displacing polymerase enzymes. The second switch involves the use of an initiating nucleic acid enzyme to cleave the BL, so that it no longer hybridises sufficiently to the DNAzyme. The now active DNAzyme can itself cleave additional BL molecules, thus triggering a cascade of release and activation of additional DNAzymes. This is referred to as the ‘DNA-only cascade’ (DOC) as it is solely driven by DNAzyme catalysis and therefore does not require the use of protein enzymes. Both cascades can be triggered by the presence of nucleic acid targets and the resulting active DNAzymes cleave reporter substrates, producing amplification of signal in real-time. Due to its protein-free composition, the DOC is considered to have particular applicability for use in POC testing and has demonstrated to be highly thermostable, perform well within complex biological media, detect multiple targets simultaneously and be amenable to attachment to solid surfaces. The range of target analytes was also extended from nucleic acids to include small molecules and metal ions, demonstrating its ability to function as a universal method for signal amplification