<p> pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein we report on the sequence truncation and secondary structure characterization ofpH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three inter-helical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides. Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides. Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. A trans-acting DNA enzyme was also developed from the minimized version ofpH6DZl, which behaves as a true enzyme with a kcat value of~1 min"1 and generates a large fluorescence signal upon catalysis. This study should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors. </p> / Thesis / Master of Science (MSc)

Pathogenic bacteria pose serious threats to public health and safety. They can cause illness, death, and substantial economic losses. The most widely used bacterial detection methods include cell culturing, antibody-based assays, and nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Unfortunately, these techniques are not well suited for point-of-care application, especially in the resource-limited regions of the world, as they require highly trained personnel to perform the test, they take a long time to complete (especially culturing), and they require sophisticated lab equipment. Thus, there is a great need for simpler, faster, and more accurate methods for bacterial detection. In this thesis, we present a simple, low-cost assay for detecting pathogenic bacteria that is based on the immobilization of a bacteria-specific RNA-cleaving DNAzyme (DNAzyme) onto a surface. If the target bacteria is present, a fluorescently labelled piece of DNA (FDNA) is released through the activity of the DNAzyme; if the target bacteria is not present, the FDNA remains attached to the surface as part of the DNAzyme construct. This method allows untrained users to determine whether a target bacteria is present by simply monitoring the fluorescence intensity in the liquid phase with a hand-held fluorimeter. The first step in this work was to experimentally evaluate different surfaces (including reduced graphene oxide and different beads) onto which the DNAzyme could be immobilized. These tests determined that agarose beads, covered with streptavidin, were ideally suited for DNAzyme immobilization. Next, we conducted a comparative evaluation of the kinetics/activity of the DNAzyme that had been immobilized onto the beads and the free DNAzyme in solution; the results of this evaluation revealed virtually identical reaction rates for the two cases, suggesting no loss of activity after immobilization. Finally, we explored how the DNAzyme sequence length influenced the assay. Specifically, we analyzed a full-length DNAzyme (Full DNAzyme) sequence and a truncated alternative (Short DNAzyme) and found that the full-length construct resulted in faster signal generation. Therefore, it was determined that the long version should be used in the assays. When coupled with a filtration step, the immobilization of biotinylated DNAzymes onto the surface of streptavidin-coated agarose beads enabled the sensitive detection of E. coli in both water samples and complex matrices, such as milk and apple juice. The bead-based assay was able to produce a strong fluorescence signal readout in as little as 2.5 min following contact with E. coli, and it was capable of achieving a detection limit of 1,000 colony-forming units (CFUs) without sample enrichment. As DNAzyme probes can be generated through in vitro selection to react to different bacteria, the RNA-cleavage based detection mechanism described in this work can be adapted for the detection of a wide range of bacterial targets. Overall, this research has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that is highly attractive for field applications, especially in resource-limited regions. / Thesis / Master of Applied Science (MASc)

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.