Advances in our ability to detect biological targets relevant to human health have come from the engineering of biological molecules into assemblies capable of performing target-induced signal generation. Such assemblies, known as biosensors, are composed of a molecular recognition element (MRE) and a signal generating transduction element. One MRE class that has received great attention in recent years is functional nucleic acids, which include DNA aptamers and DNAzymes. Since 1990, a large number of functional nucleic acids have been reported. However, broad commercial use of functional nucleic acids in applications that benefit human health is sparse. The goal of this thesis is to expand the usefulness of functional nucleic acids. The thesis is made of four projects. In the first project I developed a simple colorimetric biosensor for the detection of a toxic metal ion using a reported RNA-cleaving DNAzyme coupled with urease as the signal reporter. This is followed by a project where I developed a highly effective method for the synthesis and purification of the DNA-urease conjugate needed for the biosensor. I then turned my attention to the search for high-affinity DNA aptamers that bind VEGF-165, an important human protein found to be relevant in the progression of cancers. Given that VEGF-165 is a homodimeric protein, in my third project I looked into the suitability of reported DNA aptamers for this protein for the creation of dimeric aptamers with higher binding affinity. I examined multiple factors that may affect the successful engineering of dimeric aptamers and determined that none of the existing aptamers are compatible for creating a productive dimeric aptamer. With this finding, I made an effort to create our own aptamers for this protein target. I was able to isolate a new aptamer that appears to be an excellent candidate for creating a higher affinity DNA aptamer. Overall, my work adds to our increasing appreciation of the functional capability demonstrated by single-stranded DNA molecules. More importantly, I hope the methods I have developed and new functional DNA molecules I have generated in this thesis will continue to drive the development of the functional nucleic acid field and contribute to the health research community’s efforts to increase human longevity. / Thesis / Doctor of Philosophy (PhD)

<p>The food-borne pathogen, <em>Listeria monocytogenes</em>, is a global health concern as it has been responsible for multiple food contamination outbreaks over the past century. Current detection methods like the enzyme-linked immunoassays (ELISA), and polymerase chain reaction (PCR) take over 24 h to attain results, are costly, require specialized equipment and trained personnel. In this study we investigated the use of functional nucleic acid (FNA) to develop a rapid and cost-effective detection method for <em>L. monocytogenes</em>. We carried out in<em> vitro</em> selection in order to isolate a fluorescently labeled DNA-RNA hybrid strand that can be bound and cleaved by specific endoribonucleases (RNase) from <em>L. monocytogenes</em>. We termed these DNA-RNA hybrid strands RNase-cleaved fluorescent substrate (RFS). Since no past studies have isolated RNases from <em>L. monocytogenes</em>, we first identified the genes based on sequence similarities with well characterized RNases. We purified and characterized RNase HII, RNase III and RNase G. Since this study focused primarily on developing RFS for RNase HII, we performed an in depth <em>in vitro</em> biochemical analysis to characterize this enzyme. We found that RNase HII from <em>L. monocytogenes</em> plays an important role in DNA replication and repair. Furthermore, we obtained six sequence classes by <em>in vitro</em> selection which could interact with RNase HII. The key nucleotide regions involved with RNase HII interactions were identified. In the final study, we showed the sequences isolated by <em>in vitro</em> selection could also be used as a tool to study ribonuclease function and identify new interaction between enzyme and substrate.</p> / Master of Science (MSc)