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)

Over the last 20 years, the incidence of Clostridium difficile infection (CDI) has increased dramatically, making it one of the most common healthcare-associated infections. This has been linked to the emergence of hypervirulent C. difficile strains. Currently, cell cytotoxicity assay (CTA) and toxigenic culture are the gold-standard methods for CDI diagnosis. However, they are time-consuming and labour-intensive. Other methods, like enzyme immunoassays (EIAs) and nucleic acid amplification-based tests (NAATs), allow for rapid testing but have poor sensitivity and/or specificity. Additionally, most of these methods target toxins or their associated genes and are unable to discriminate between epidemic and non-epidemic strains. The work described in this dissertation aims to develop easy-to-use and reliable biosensors for C. difficile, with a particular focus on epidemic strains of C. difficile. The development of the in vitro selection technique has allowed for the discovery of a big array of functional DNA, with excellent ability in both target recognition and enzymatic catalysis. My key interest is to employ functional DNA molecules as target recognition elements to develop colorimetric biosensors for C. difficile detection. The first research project aimed to develop a colorimetric detection platform that can be coupled with functional DNA molecules to achieve hypersensitive detection of different targets. This test should be easy-to-use, have broad target applicability and not require expensive equipment. To do so, a colorimetric biosensing platform was created, which takes advantage of the signal amplification ability of rolling circle amplification (RCA) and the simplicity of the classic litmus test. In the presence of the target of interest, RCA will be triggered, and the biotinylated RCA products can hybridize a number of the urease-labelled single-stranded DNA and immobilize urease onto magnetic beads through streptavidin-biotin interactions. The urease can then be used to hydrolyze urea, resulting in significant pH elevation, that can be detected easily using a litmus test. To prove the concept, we have demonstrated that this platform can be employed to visually detect thrombin and platelet-derived growth factor (PDGF) with high sensitivity, by coupling it with an anti-thrombin aptamer and an anti-PDGF aptamer, respectively. We have also shown that the biosensing platform can be incorporated into simple paper-based devices. The second project focuses on the development of a colorimetric DNA detection method for epidemic strains of C. difficile that utilizes both the polymerase chain reaction (PCR) and the litmus test. The strategy makes use of a modified set of primers for PCR to facilitate ensuing manipulations of resultant DNA amplicons: their tagging with urease and immobilization onto magnetic beads. The amplicon/urease-laden beads are then used to hydrolyze urea, resulting in an increase of pH that can be conveniently reported by a pH-sensitive dye. We have successfully applied this strategy for the detection of two hypervirulent strains of C. difficile, which are responsible for the recent increase in the global incidence and severity of C. difficile infections. Furthermore, the viability of this test for diagnostic applications is demonstrated using clinically validated stool samples from C. difficile infected patients. The goal of the third project was to isolate RNA-cleaving fluorogenic aptazymes (RFAs) targeting an epidemic strain of C. difficile. Four classes of RFA probes were derived using in vitro selection approach where a random-sequence DNA library was reacted with a crude extracellular mixture (CEM) derived from the epidemic C. difficile strain BI/027/NAP1, coupled with a subtractive selection strategy to eliminate cross-reactivities to unintended C. difficile strains and other bacterial species. The isolated RFDs can be used together to generate specific cleavage patterns for strain-specific identification of C. difficile. Lastly, the final project was to characterize a novel RFA probe (RFA13-1) that was isolated unintentionally using in vitro selection. By using CEM prepared from C. difficile glycerol stock contaminated by Klebsiella aerogenes as the positive target for in vitro selection, we isolated a remarkably active RFA probe, RFA13-1, targeting K. aerogenes. Further studies demonstrated that RFA13-1 could be activated by CEM prepared from several bacteria from the Enterobacteriaceae family. Moreover, the molecular target of RFA13-1 has been identified, which is ribonuclease I. RFA13-1 showed high sensitivity and specificity towards RNase I and could be employed as a tool to study RNase I functions and to detect RNase I or RNase I-containing bacteria. In summary, I have investigated novel strategies for building a biosensor that is capable of discriminately detecting epidemic strains of C. difficile. I hope that my work can take us one step closer towards the development of easy-to-use and reliable biosensors that can be used in the clinical diagnosis of CDI. / Thesis / Doctor of Philosophy (PhD)

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)

RNA-cleaving DNAzymes (RCDs) are a class of functional nucleic acids that can bind various targets ranging in size from small molecules to large proteins, which results in activation of cleavage activity. The activation of RCDs results in the cleavage of a ribonucleotide site in an otherwise all-DNA substrate, leading to two cleavage fragments. In this work, a previously identified DNAzyme that binds to a protein biomarker endogenous to Helicobacter pylori (J99) crude extracellular matrix was evaluated for coupling to an isothermal amplification method termed rolling circle amplification (RCA) as a way to improve the originally reported detection limit. Three RCD constructs were designed with the goal of generating a cleavage fragment that could act as a primer to initiate RCA. The first method used the original HP DNAzyme, which liberated a short cleavage fragment that could be used as a primer. However, the primer fragment was rapidly digested by the bacterial matrix, preventing RCA. A second method evaluated use of a circularized substrate and separate RCD to generate a primer, however this system was not capable of generating a cleavage fragment. A final method redesigned the original RCD to move the substrate region from the 3’ to the 5’ end of the RCD, causing the longer RCD-containing fragment to be the primer for RCA. In this case, target-triggered cleavage was observed and the resulting primer was sufficiently resistant to digestion to allow its use as a primer for RCA. Preliminary characterization of the rearranged RCD showed that it retained selectivity similar to the original RCD, but that the cleavage rate was slower. In addition, the RCA based reaction, while successful, did not produce improved detection sensitivity relative to unamplified assays. Methods to further improve RCA performance are discussed for future work. / Thesis / Master of Science (MSc)

This thesis describes the use of functional nucleic acid (FNA) superstructures entrapped within monolithic macroporous sol–gel-derived silica for solid-phase flow-based sensing of small molecules and macromolecular proteins. The work described herein overcomes a long-standing issue with entrapment of biomolecule into sol–gel-derived materials; the mesoporous pore morphology required to retain entrapped biomolecules prevents detection of large analytes as these can’t access the entrapped species. It is shown that large DNA superstructures can be produced through rolling circle amplification of a functional nucleic acid, resulting in concatemeric FNA species with dameters of several microns. Such species can be entrapped within macroporous sol-gel derived materials with micron-sized pores with minimal leaching, thus allowing for detection of a wide range of molecules, including biomolecules. Optimal materials for entrapment of FNA superstructures was achieved using a high-throughput material screening method, which minimized biomolecule leaching while maintaining FNA activity. Using an optimized material, concatemeric aptamer superstructures were entrapped within macroporous monolithic columns for flow-based detection of small molecules and proteins, extending the range of analytes that can be analyzed using biohybrid monolithic columns. Preliminary studies on the formation and properties of a DNAzyme superstructure for detection of E. coli detection were also performed, which provided valuable information on factors that must be controlled to allow reproducible fluorescence-based detection of E. coli using the crude intracellular matrix as the target. / Thesis / Master of Science (MSc)

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)

Steady advancements in diagnostics over the past century have propelled the world of medicine into the more advanced era of preventative medicine, an era with a resoundingly clear message: early detection can save lives. For patients who suffer from either pancreatic cancer or malignant hyperthermia susceptibility, early or preoperative diagnosis, respectively can save lives and minimize morbidity and mortality, in addition to offering cost-savings to hospitals and healthcare systems. Fortunately, significant progress have been made in the fields of metabolomics and biomarker identification. Given the benefits carried by serum biomarkers as targets of screening and diagnostic tool development, we applied functional nucleic acid technology and in vitro selection directly in whole human serum to search for disease-specific biomarkers and associated detection probes without a priori knowledge of the biomarkers pursued. This endeavour simultaneously serves as a proof-of-concept study to establish whether in vitro selection can be successfully performed in human serum. We specifically focused on the derivation of RNA-cleaving DNAzymes (RCD) through in vitro selection, or SELEX (systemic evolution of ligands through exponential exposure). DNAzymes constructed with a fluorogenic signalling molecule were incubated with human serum with the goal of identification of a functional nucleic acid probe capable of detecting the presence of a disease-specific biomarker. Two independent protocols have been designed and executed for the identification of DNAzyme sequences capable of detecting pancreatic cancer and malignant hyperthermia susceptibility, respectively. The first exploration was performed in serum obtained from cancer patients, with the goal of identifying DNAzymes capable of distinguishing pancreatic cancer from other cancer types. To do so, we employed in vitro selection, Next-Generation Sequencing, and bioinformatic analysis. We successfully demonstrated the feasibility of performing in vitro selection with DNAzymes in human serum, evidenced by distinct round-to-round enrichment of a DNA library towards the identification of DNAzymes capable of detecting pancreatic cancer. Additionally, we isolated two DNAzymes capable of distinguishing pancreatic cancer serum from healthy patient serum in fresh collected serum samples. Based on the positive results gathered in the pancreatic cancer in vitro selection project, we subsequently endeavoured to replicate the demonstrated feasibility of performing in vitro selection in human serum. By selecting malignant hyperthermia as the pathology investigated, we simultaneously sought to diversify the scope of DNAzyme detection by establishing whether successful DNAzyme selection can be achieved in a non-acute disease state. Thus, the second exploration was performed in serum obtained from patients who underwent evaluation for malignant hyperthermia susceptibility using the gold-standard caffeine-halothane contracture test. The goal of this project rested on the identification of DNAzymes capable of distinguishing malignant hyperthermia susceptibility in serum and approximating the performance of the gold standard test. We successfully isolated four DNAzyme candidates which demonstrated clinically relevant thresholds of sensitivity and specificity following thorough sensitivity and specificity analysis. In doing so, we once again demonstrated the ability to perform in vitro selection in human serum. Given the complexity of molecular interactions observed over the course of two in vitro selection protocols in human serum, it became clear that distinguishing meaningful target-mediated interactions from non-specific interactions would require advanced bioinformatic analysis. Consequently, using principles of computational biology, we performed a deep exploration of Next-Generation Sequencing results obtained from sequencing our recovered DNA libraries to extract additional data that would inform on the next required steps required to identify a DNAzyme specific for the pathology pursued. In doing so, we identified a two-step method to evaluate the progress of the in vitro selection protocol undertaken, and offered a systematic approach for choosing candidate sequences to undergo further testing based on promising performance in silico. Using this approach, we successfully identified a DNAzyme sequence capable of acting as a general cancer detection probe, with promising potential for diagnostic application. Ultimately, this thesis serves as a feasibility study of a novel approach to both in vitro selection and biomarker identification technique by combining the latest nanotechnology techniques with clinical data and real patient serum samples, and advanced computational biology tools. Despite the inability to identify a highly sensitive and specific DNAzyme capable of advancing towards biosensor construction, several important strides and lessons have been acknowledged, establishing the feasibility of performing in vitro selection in human serum, and outlining strategies for addressing and anticipating challenges with this technique. The hope is for this work to inspire and inform future efforts to apply functional nucleic acid technology to solve current gaps in both the diagnostic and therapeutic branches of medicine, and with the help of computational biology continue to bridge the gap between basic science and clinical medicine. / Dissertation / Doctor of Philosophy (PhD)