The urokinase receptor (uPAR) plays a central role in metastatic process. It???s evident uPAR is overexpressed across a variety of tumour cells and leads to the increased aggressiveness and poor prognosis of cancer. Inhibition of uPAR expression can block metastatic potential in many tumours. In addition, besides uPA, there are several other proteins which have been confirmed to interact with uPAR, such as vitronectin and integrins. These interactions also contribute to signal transduction and the functions of uPAR complex. Therefore, downregulation of uPAR expression by targeting uPAR mRNA or protein, or by regulating the uPAR partners would be potential therapeutic strategies for prevention of cancer metastasis. There are two main aspects contained in this thesis. Firstly, three specific DNAzymes targeting uPAR mRNA were designed to downregulate uPAR expression in vitro and their effects to decrease cancer cell invasion studied in a human osteosarcoma cell line Saos-2. The results showed that two of them (Dz483 and Dz720) cleaved uPAR transcript in vitro with high efficacy and specificity and the Dz720 inhibited uPAR protein levels by 55% in Saos-2 cells. Besides, the Dz720 significantly suppressed Saos-2 cell invasion using an in vitro matrigel assay. Secondly, two potential uPAR partners from yeast two-hybrid screening, a heat shock protein MRJ and an anti-apoptosis protein HAX-1, were characterised and their functions binding with uPAR investigated. The interactions were confirmed by co-immunoprecipitation, GST-pull down assay and confocal microscopy in cancer cells. In addition, there was a 50% increase in cell adhesion after transfection with MRJ. This increase in adhesion is dependent on the uPAR/full length MRJ interaction as cells transfected with the mutant construct containing only N-terminal region or C-terminal region of MRJ had no increase in cell adhesion. The observed increase in adhesion to vitronectin by MRJ was also blocked by an anti-uPAR domain I antibody suggesting that the induced adhesion is at least in part contributed by uPAR on the cell surface. Together, the identification of both MRJ and HAX-1 as uPAR interactors provides further insight into the intricate relationship between uPAR and other proteins which may develop potential approaches for cancer therapy.

The urokinase receptor (uPAR) plays a central role in metastatic process. It???s evident uPAR is overexpressed across a variety of tumour cells and leads to the increased aggressiveness and poor prognosis of cancer. Inhibition of uPAR expression can block metastatic potential in many tumours. In addition, besides uPA, there are several other proteins which have been confirmed to interact with uPAR, such as vitronectin and integrins. These interactions also contribute to signal transduction and the functions of uPAR complex. Therefore, downregulation of uPAR expression by targeting uPAR mRNA or protein, or by regulating the uPAR partners would be potential therapeutic strategies for prevention of cancer metastasis. There are two main aspects contained in this thesis. Firstly, three specific DNAzymes targeting uPAR mRNA were designed to downregulate uPAR expression in vitro and their effects to decrease cancer cell invasion studied in a human osteosarcoma cell line Saos-2. The results showed that two of them (Dz483 and Dz720) cleaved uPAR transcript in vitro with high efficacy and specificity and the Dz720 inhibited uPAR protein levels by 55% in Saos-2 cells. Besides, the Dz720 significantly suppressed Saos-2 cell invasion using an in vitro matrigel assay. Secondly, two potential uPAR partners from yeast two-hybrid screening, a heat shock protein MRJ and an anti-apoptosis protein HAX-1, were characterised and their functions binding with uPAR investigated. The interactions were confirmed by co-immunoprecipitation, GST-pull down assay and confocal microscopy in cancer cells. In addition, there was a 50% increase in cell adhesion after transfection with MRJ. This increase in adhesion is dependent on the uPAR/full length MRJ interaction as cells transfected with the mutant construct containing only N-terminal region or C-terminal region of MRJ had no increase in cell adhesion. The observed increase in adhesion to vitronectin by MRJ was also blocked by an anti-uPAR domain I antibody suggesting that the induced adhesion is at least in part contributed by uPAR on the cell surface. Together, the identification of both MRJ and HAX-1 as uPAR interactors provides further insight into the intricate relationship between uPAR and other proteins which may develop potential approaches for cancer therapy.

The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world’s population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient’s sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.

<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn²⁺, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p> / Doctor of Philosophy (PhD)

While the Canadian food supply is among the healthiest in the world, almost 4 million (1 in 8) Canadians are affected by food-borne illnesses, resulting in 11,600 hospitalizations and 238 deaths per year. Microbial pathogens are one of the major causes of foodborne sicknesses that can grow in food before or following packaging. Food distribution is an important part of the food processing chain, in which food supplies are at a higher risk of contamination due to lack of proper monitoring. Among myriad of research around biosensors, current devices focusing on packaged food monitoring, such as leakage indicators or time temperature sensors are not efficient for real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging and capable of tracing the presence of pathogens, are of great interest to ensure food safety. This work involves developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which report the presence of a target bacterium in food or water samples by generating a fluorescence signal that can be detected by simple fluorescence detecting devices. The covalently-attached DNA probes generate the signal upon contact with the target bacteria with as low as 103 CFU/mL of Escherichia coli in meat and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to detecting pathogens on packaged food or drinking bottles, these surfaces have the potential to be used for a variety of other applications in health care settings, environmental monitoring, food production chain, and biomaterials like wound dressing. / Thesis / Master of Science (MSc) / Microbial pathogens can grow in food following packaging and preceding consumption. Current biosensors are not efficient for post-packaging real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging, tracing the presence of pathogens, are much useful to ensure the food safety. Here we report on developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which generate a fluorescence signal in the presence of a target bacterium in food or water samples. The covalentlyattached DNA probes can detect as low as 103 CFU/mL of Escherichia coli in meat, sliced apple and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to pathogen monitoring in packaged food or drinking bottles, these surfaces are promising for a variety of other applications in health care settings, environmental monitoring, and biomaterials like wound dressing.

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)

Formés à partir de brins d’ADN ou d’ARN riches en guanines, les quadruplexes résultent de l’empilement de tétrades de guanines constituées chacune par l’auto-assemblage dans un même plan de quatre guanines, stabilisées entre elles par un réseau de liaisons hydrogènes. En s’inspirant de cet édifice naturel, il est présenté au long de ce manuscrit de thèse la synthèse et l’étude de molécules de type TASQ (pour template-assembled synthetic G-quartet) hydrosolubles capables de former de manière intramoléculaire une tétrade de guanines synthétique : les DOTASQ, le PorphySQ et le PNADOTASQ. La première application développée pour ces composés est le ciblage des quadruplexes d’ADN et d’ARN, présents dans des régions clefs du génome (télomères, promoteurs d’oncogènes) et du transcriptome (5’-UTR et TERRA), et dont la stabilisation par un ligand pourrait ouvrir de nouvelles perspectives en terme de thérapie antitumorale ciblée. Les résultats in vitro sont présentés et permettent de démontrer que les TASQ hydrosolubles développés sont des composés offrant une bonne sélectivité pour les quadruplexes mais surtout une excellente sélectivité grâce à un mode d’action bioinspiré basé sur une reconnaissance biomimétique. La seconde application mise au point est l’utilisation des TASQ comme catalyseurs pour des réactions de peroxydation : leur architecture même leur permet de mimer l’activité catalytique de l’ADN (ou DNAzyme) ainsi que celle de protéines (enzyme) comme la horseradish peroxidase. Ce processus est dépendant de la formation intramoléculaire de la tétrade de guanines synthétique et ouvre de nombreuses perspectives en terme d’utilisation en biologie ainsi qu’en nanotechnologie. / Natural G-quartets, a cyclic and coplanar array of four guanine residues held together via Hoogsteen H-bond network, have recently received much attention due to their involvement in G-quadruplex-DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events (chromosomal stability, regulation of gene expression). Besides this, synthetic G-quartets, which artificially mimic native G-quartets, have also been widely studied for their involvement in nanotechnological applications (i.e. nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G-quartets, also named template-assembled synthetic G-quartet (TASQ), have been more sparingly investigated, despite a technological potential just as interesting.In this way, we designed and synthesized three series of innovative hydrosoluble TASQ: DOTASQ (for DOTA-Templated Synthetic G-Quartet), PorphySQ (containing a porphyrin template) and the most effective PNADOTASQ where PNA-guanine arms replace native DOTASQ alkyl-guanine arms. We report herein the results of both DNA and RNA interactions (notably their selective recognition of quadruplex-DNA according to a bioinspired process) and peroxidase-like hemin-mediated catalytic activities (either in an autonomous fashion as precatalysts for TASQzyme reactions, or in conjunction with quadruplex-DNA as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes Tuberculosis (TB) and is responsible for an average of 1.5 million deaths annually. Although a treatment regimen does exist, Multi-Drug Resistant (MDR-TB) and eXtremely Drug Resistant (XDR-TB) TB strains are becoming a more prevalent concern partly due to failure of patient compliance with the current six to nine month drug treatment regimen. The current diagnostic methods are not able to identify these MDR and XDR-TB strains efficiently therefore more effective point-of-care (POC) diagnostics and drug susceptibility testing (DST) are urgently needed to detect drug resistance and facilitate prompt, appropriate treatment plans. In order to detect TB and efficiently identify drug resistance, this project seeks to develop a novel diagnostic technology based on deoxyribozyme (DNAzyme) sensors. The overall goal of this project is to create an assay which combines Polymerase Chain Reaction (PCR) and DNAzymes to identify drug resistance conferring Single Nucleotide Polymorphisms (SNPs). To safely test the ability of DNAzyme sensors to detect SNPs indicative of multi-drug resistant TB, we have constructed a panel of drug resistant (drugR) nonpathogenic M. bovis BCG. We have designed a multiplex PCR that amplifies 6 chromosomal regions of the genome necessary for the species specific detection of TB and determination of a drug susceptibility profile based on the presence of SNPs. To improve the sensitivity and selectivity of the detection and DST of Mtb, we have designed and optimized DNAzyme sensor assays combined with multiplex PCR analytes that will enable the rapid, POC detection of drug resistance. This work aims to develop novel tools for the prompt and specific diagnosis of TB allowing for the implementation of an iv effective treatment regimen that will ultimately lessen transmission and control the emerging global threat of MDR and XDR-TB.

Despite the increasing number of electrochemical and photoelectrochemical biosensors reported in the research literature, few have achieved success outside of a laboratory setting. This can partly be attributed to accessibility issues with commercially available readout instruments. Consequently, low-cost and portable readout instruments have been developed by researchers, but these devices fail to address other key compatibility and accessibility challenges. Much like the commercial systems, these devices are not natively compatible with multiplexed signal assays consisting of two or more working electrodes, cannot control optical excitation sources for photoelectrochemical biosensing, nor can they interface with auxiliary instruments such as heaters and electromagnets. To this end, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system for point-of-care biosensing. Our readout system can perform standard voltammetric techniques and is capable of synchronously controlling an optical excitation source to support photoelectrochemical biosensing. This device is compatible with standard three-electrode assays as well as dual signal assays with two working electrodes. We have also created a portable sample heater that can be controlled by this readout system to facilitate on-site sample heating and have also integrated a portable electromagnet to perform away-from-lab magnetic manipulation. / Thesis / Master of Applied Science (MASc) / Early and prompt detection of disease biomarkers is crucial in order to develop effective disease management strategies. Unfortunately, many gold-standard diagnostic techniques for infectious diseases, cancers, heart diseases, among other conditions prove to be time-consuming, costly, and reliant on trained professionals in a laboratory setting. Electrochemical and photoelectrochemical detection are two sensing modalities that show promising potential for point-of-care applications, as they are easily miniaturized, inexpensive, and can be used to detect both the presence of and the amount of analyte present. However, up until now, these sensing modalities have mostly been confined to research settings. To expedite the commercialization of such sensors and to facilitate their translation to point-of-care diagnostics, we have developed a low-cost smartphone-operated electrochemical and photoelectrochemical readout system. Through the integration of peripheral instruments including a sample heater, electromagnet, and optical excitation source, this system is compatible with a number of different biosensors.

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.