Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, accounting for over 600,000 deaths annually. The mortality rate of CRC can be significantly reduced if it is detected early, suggesting the importance of cancer screening in CRC management. Currently, colonoscopy is the gold standard for CRC diagnosis as it is accurate and reliable. However, it is an invasive procedure that is associated with risks of complications, which contributes to the lack of patient compliance in colonoscopy screening. Other noninvasive detection methods suffer from poor sensitivity and specificity. Thus, there remains a great demand for the development of a noninvasive and accurate test for CRC diagnosis. Recently, studies using next-generation sequencing techniques have revealed compositional changes in the intestinal microbiome associated with CRC, implicating the possibility of using fecal microbiome as potential diagnostic markers. Specifically, the level of the gram-negative bacterium, Fusobacterium nucleatum, has been shown to be elevated in CRC patients compared to healthy controls. The work described in this thesis aims to develop unique RNA-cleaving DNAzymes that can distinguish between healthy and CRC stool microbiomes. RNA-cleaving DNAzymes are single stranded DNA molecules that are extensively used as analytical tools for metal ion sensing and bacterial detection. We conducted an in vitro selection experiment and isolated a F. nucleatum-responsive RNA-cleaving DNAzyme sensor, named RFD-FN1, that is activated by a heat stable protein marker by this bacterium. RFD-FN1 is highly specific for F. nucleatum and it has a limit of detection of 107 CFU/mL without culture and a single cell when cultured for 36 hours. The discovery of this novel molecular probe for F. nucleatum presents an important step forward towards the development of a novel DNAzyme-based detection method for colorectal cancer. / Thesis / Master of Science (MSc)

The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.

The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.

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)