Ineffective bacterial monitoring in water systems represents a danger to public health and can result in costly disease outbreaks. Of interest is Legionella pneumophila, a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. The Center for Disease Control stated that reported cases of Legionnaires’ disease have quadrupled since 2000 and ranks L. pneumophila as the number one cause of waterborne disease outbreaks in the United States. This threat is expected to increase given an aging population who are more susceptible to L. pneumophila infection and rising global temperatures that can promote L. pneumophila growth. Presently, Public Health agencies recommend bacterial culturing for the detection of L. pneumophila in environmental samples, however, this process can take up to ten days to complete. Consequently, there is a delay between sample collection and subsequent L. pneumophila detection, creating an opportunity for a Legionnaires’ disease outbreak to occur. There is a great need to develop a field-appropriate device that can provide early-stage detection of L. pneumophila in water as a means of mitigating Legionnaires’ disease outbreaks. We propose the use of DNAzymes for the development of such a device. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have successfully isolated an RNA-cleaving fluorescent DNAzyme (RFD) specific for the detection of L. pneumophila using in vitro selection. Thorough characterization of the DNAzyme has revealed key structural features influencing kinetics, specificity and sensitivity. In addition, the ability of the DNAzyme to function in cooling tower water, and conservation of the DNAzyme target across Legionella bacteria, has been investigated. In the future we plan to incorporate this RFD into a field-appropriate paper-based device which would play a key role in managing infectious diseases and preventing large-scale outbreaks. / Thesis / Master of Health Sciences (MSc) / Legionella pneumophila is a deadly water-borne bacterial pathogen that causes Legionnaires’ disease - a severe form of pneumonia. Numerous Legionnaires’ disease outbreaks have occurred, with the most common source of exposure to L. pneumophila coming from contaminated cooling towers. Presently, bacterial culturing is used to determine if a cooling tower is contaminated with L. pneumophila, however this process can take up to 10 days to complete. To address this delay, we plan to develop a rapid paper-based test for L. pneumophila detection in cooling tower water using DNAzymes. DNAzymes are small, catalytically-active single-stranded DNA molecules that demonstrate target-specific enzymatic activity. We have isolated a DNAzyme that can specifically detect L. pneumophila and characterized its properties. In the future we plan to incorporate this DNAzyme into a field-appropriate paper-based test which would play a key role in managing Legionnaires’ disease outbreaks.

The development of the in vitro selection technique permits the creation of synthetic DNA molecules with ligand-binding capabilities (DNA aptamers), or abilities to catalyze chemical reactions (DNAzymes), or both (aptazymes). Significant research efforts in this field over the past two decades have led to the creation of a large array of DNA aptamers and DNAzymes and ever-increasing interests in taking advantage of these molecular species for diverse applications. One area of remarkable potential and development is the exploration of functional DNA molecules for bioanalytical applications. The work described in this dissertation aims to pursue innovative concepts and technologies that expand utility of functional DNA molecules for biosensing applications. I have focused on two functional DNA species: RNA-cleaving DNAzymes and protein-binding DNA aptamers. My key interest is to develop simple but effective colorimetric assays that employ these functional DNA molecules and to establish an effective strategy that makes functional DNA biosensors highly functional in biological samples. / Thesis / Doctor of Philosophy (PhD)

Life would not exist in the absence of catalysis. The “RNA World” model for the origin of life hinges on the capabilities of ribonucleic acid to encode information and perform catalysis (i.e. self-replication). Previously, functional nucleic acids such as ribozymes and deoxyribozymes (DNAzymes) have been isolated using the process of in vitro selection. This method is typically performed by isolating a catalytically active molecule from a large random library, with the assumption being that active molecules are already present in the pool and this method filters them from inactive molecules. However, in vitro selection has never been used to show that a molecule can be evolved from an inactive to an active catalyst. Here we show that the properties of DNA can be exploited to act as a proxy system for the origins of biotic chemistry by isolating a functional catalyst from a previously non-catalytic sequence. This project employs a novel perspective; rather than a random library, a known, non-functional sequence is utilized. Using in vitro selection, this known sequence is gradually evolved into a functional catalyst by solely allowing the existence of sequences that acquire mutations which enhance their function. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of molecular evolution. Evolving a catalyst from a non-catalyst gives insight into the comprehension of how life originated. This project demonstrates that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today. / Thesis / Master of Science (MSc)

Escherichia coli (E. coli) is a gram-negative bacteria found in the intestinal system of humans that can also contaminate food, drinking water, as well as lakes and rivers. While not all strains are pathogenic, some including O157:H7 can cause severe illness. Conventional methods of detecting E. coli contamination in water samples often have limitations for on-site testing applications, which can include their slow detection time or need for expensive laboratory equipment. While several fluorescent biosensors for the detection of E. coli have been developed in the Didar lab, there is increased interest in colourimetric biosensors whose signal can be interpreted with the naked eye. This thesis will describe the development and performance of a hydrogel biosensor, that is made of polyacrylamide chains crosslinked by an E. coli detecting Deoxyribozyme (DNAzyme) and its substrate. In the presence of E. coli, the DNAzyme cleaves its substrate and crosslinking breaks down, resulting in the visible dissolution of the hydrogel. Paired with the use of bacteriophage induced cell lysis to amplify the target protein, detection sensitivity to the order of 10^1 CFU/mL has been achieved using this platform with an incubation time of 18 hours. A convolutional neural network (CNN) trained on optical images of the platform was able to classify samples as contaminated or uncontaminated with a validation accuracy of over 93%. / Thesis / Master of Applied Science (MASc) / Microbial contamination of water sources including surface water, groundwater, and drinking water can pose risks to human health. One bacterial species that can sometimes contaminate these sources is Escherichia coli (E. coli). To determine if E. coli is present in a water sample, it often needs to be sent to a laboratory for testing, which can be time consuming and inconvenient. Therefore, researchers are working to develop new sensors that are able to detect E. coli from water samples, ideally being simple enough to use that testing could be done right away and without sending the sample to another location. In this research project, we have developed a new biosensor that can detect E. coli in water samples. To use the sensor, a water sample is added onto a small red gel in a tube, and this gel breaks apart if E. coli is present in the sample.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

The functionality of nucleic acids beyond genetics has attracted more attention over the past decades. Functional nucleic acids (FNA), including aptamers and nucleic acid-based enzymes, are well-known for their target binding and reaction catalysis abilities. FNA can be obtained through a technology called in vitro selection, which allows the isolation of customized FNA for various applications. In particular, FNA have received much interest in biosensing application. Their wide range of sensing targets, intrinsic stability, and high specificity have qualified them as the molecular recognition element in biosensors. This thesis explored the utilization of FNA to tackle real-world biosensing challenges, especially for pathogenic bacteria detection. The first project aimed to make the most use of in vitro selection to derive FNA that can meet the requirements of terminal applications. A few feasible approaches were proposed based on lessons from Mother Nature and validated by innovative scientist pioneers. In the second research project, I characterized an RNA-cleaving DNAzyme for Clostridium difficile infection diagnosis. This DNAzyme displayed high sensitivity and specificity for clinical C. difficile strains, making it a competitive candidate for a potential point-of-care diagnostic tool. In the next chapter, I incorporated a Legionella pneumophila-responsive RNA-cleaving DNAzyme into a bead-based assay for practical on-site detection. This assay exhibited a high stability and functionality in the cooling tower water samples, the real-world application environment. The following chapter was to optimize this assay further with a coupled rolling circle amplification strategy. This additional amplification speeded up the detection process, improved the limit of detection, and enabled the colorimetric results that are observable to the naked eye. These research aimed to advance the practical applications of FNA as key components in biosensors. I hope readers find this thesis insightful and inspirational for the development of the FNA field. / Thesis / Doctor of Philosophy (PhD)

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)

It is now widely known that some nucleic acid molecules, either DNA or RNA, are capable of forming intricate three-dimensional structures and carrying out functions of molecular recognition and catalysis. Most of known functional nucleic acids are isolated from DNA or RNA pools with random sequences using the technique of in vitro selection. With intensive research for the past three decades, a variety of functional nucleic acids have been discovered and examined for potential applications. The general objective of this thesis is to expand the repertoire of functional nucleic acids via new in vitro selection experiments and pursue their biosensing applications. I started by asking the question of whether it is possible to develop a new kind of functional nucleic acids: chimeric RNA/DNA substrates that have high activity for ribonuclease H2 from the important bacterial pathogen Clostridium difficile but much reduced activity towards the same enzymes from other bacterial species. The key rationale behind pursuing these special functional nucleic acids is my hypothesis that these molecules can eventually be developed into useful biosensors for diagnosing Clostridium difficile infection. For this reason, in my first project, I applied the in vitro selection technique to a random-sequence DNA pool, obtained several highly selective chimeric RNA/DNA substrates, and carried out in-depth analysis of their reactivities and their structural properties. During this study, I accidentally discovered a family of highly guanine-rich DNA molecules that are able to form an unusual guanine-quadruplex structure in 7 molar urea, a strong denaturing condition for nucleic acid structures. This discovery constitutes a novel observation and therefore, in my second project, I fully characterized the sequence and structural properties of these special DNA molecules and established the conditions that allow these molecules to create stable structures in 7 molar urea. I then got interested in devising a unique application to take advantage of the urea-resistant property exhibited by these molecules. Towards this end, in my third project, I used one such DNA molecule to set up a DNA detection method capable of detecting single nucleotide polymorphism in very long DNA sequences, a desired application that has never been demonstrated before. The findings made in these projects contribute to the ever-growing appreciation of functional capability and practical utility of nucleic acids. / Thesis / Doctor of Philosophy (PhD)

The need for a non-invasive, accurate, easy-to-use, and cost-effective colorectal cancer (CRC) detection device is apparent in the low survival rates seen in late-stage diagnoses. Once CRC has progressed past stage I, the 5-year survival rate drops significantly, and treatment options become less favourable. The best way to treat CRC is to catch it early. The development of an RNA-cleaving fluorogenic DNAzyme (RFD) holds the potential to remediate this deficiency. A DNAzyme, called RFD-FN1, was identified from a synthetic random-sequence DNA library to selectively bind to an unknown target associated with Fusobacterium nucleatum, which has been found to be overabundant in pre- and cancerous colorectal tissue and stool. Target recognition by the DNAzyme induces the cleavage of a fluorogenic substrate and generates a fluorescent signal to indicate the presence of the bacterium. This thesis outlines the efforts made towards functionalizing the F. nucleatum-responsive probe in stool samples to create a non-invasive screening test. RFD-FN1 is selective towards a heat-stable F. nucleatum protein, but its limit of detection is only 10^7 CFU/mL. Although able to detect spiked concentrations of F. nucleatum cells in processed stool samples, the use of heat, filtering, centrifugation, antibiotics, culturing or serial dilutions are not sufficient to detect the F. nucleatum that is naturally present in the diseased samples. Experiments designed to enrich the target concentration revealed that the target is not produced consistently in any growing condition tested. Size exclusion chromatography and mass spectrometry analysis identified five potential targets that RFD-FN1 may be responding to. Three candidate targets were cloned and purified, but they failed to induce RFD-FN1’s activity. Due to the COVID-19 pandemic, the purification of the final two proteins was not completed. Purifying the two candidate targets and testing their ability to induce RFD-FN1 represents future research efforts. If the target for the DNAzyme is confirmed, a reselection for a more sensitive DNAzyme, that can function in human stool, can be attempted. / Thesis / Master of Health Sciences (MSc)