<p>Cytochrome c (Cyt c) is a heme-containing protein that is a component of the electron transport chain as well as the mitochondrial apoptotic pathway. It is released from the mitochondrial intermembrane space to the cytosol during apoptosis and is also thought to be a biomarker for cancer and liver disease. Therefore, an efficient Cyt c biosensor would be a very useful tool for studying apoptosis. Here we show the process of development of Cyt c-dependent aptazymes, derived by <em>in vitro </em>selection. These aptazymes consist of 3 components: 1) a substrate with a cleavage site that consists of a single ribonucleotide flanked by a fluorophore and quencher; 2) a DNAzyme (catalytic DNA) motif capable of cleaving the substrate; 3) an aptamer, a short piece of single- stranded DNA that can specifically bind Cyt c. When Cyt c is absent, the aptamer occludes the catalytic core of the DNAzyme and the fluorophore of the intact substrate is quenched. However, when Cyt c is present, the aptamer binds Cyt c, allowing the DNAzyme to cleave the embedded ribonucleotide, separating the fluorophore and quencher, resulting in a fluorescent signal. Simulations of <em>in vitro </em>selection of Cyt c- dependent aptazymes were also performed. The simulations revealed several methods that can improve the success rate of future <em>in vitro </em>selections of aptazymes.</p> <p>Further analysis of the previously derived DNAzyme DEC22-18 was also performed. A detailed understanding of this DNAzyme will allow it to be developed into a biosensor.</p> / Master of Science (MSc)

The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)