A galectina-1 (Gal1) é uma lectina, altamente conservada, que reconhece ?- galactosídeos, e está envolvida na regulação da tolerância da imunidade celular e na homeostase. Dados da literatura mostram que esta lectina endógena é amplamente expressa em locais de inflamação e na tumorigênese, participando diretamente dos processos de adesão celular, crescimento tumoral, metástase e angiogênese, ressaltando a relevância de sua detecção em amostras biológicas, e sugerindo que a inibição dirigida da Gal1 pode resultar em benefícios no tratamento de distúrbios inflamatórios e em novas estratégias terapêuticas antitumorais. Entretanto, ainda são escassos os dados sobre inibidores de Gal1 com real impacto terapêutico no bloqueio da atividade biológica dessa lectina. Os aptâmeros são oligonucleotídeos de cadeia simples (DNA ou RNA), que podem se ligar a uma vasta diversidade de alvos, tais como íons, peptídeos, proteínas, moléculas orgânicas e inorgânicas, com alta afinidade e especificidade. Os aptâmeros são selecionados a partir de bibliotecas com sequências randômicas de oligonucleotídeos fita simples (ssDNA) constituídos por uma região central variável, flanqueada por duas regiões de interação com primers para amplificação das sequências via PCR. Esse processo de seleção é denominado de Evolução Sistemática de Ligantes por Enriquecimento Exponencial (SELEX). Neste trabalho foram selecionados e caracterizados aptâmeros de DNA que se ligam a Gal1 humana recombinante e inibem sua atividade lectínica. O processo de seleção dos aptâmeros foi feito através de uma variação da metodologia SELEX, desenvolvida neste trabalho e aqui denominada de \"single vial selection\" (SVS), na qual todas as etapas de seleção dos aptâmeros ocorreram em um único recipiente, de forma rápida e eficiente, evitando etapas cromatográficas, que geralmente são utilizadas no SELEX. Análises com a técnica de termoflúor (TSA) e espectroscopia de fluorescência intrínseca do triptofano permitiram confirmar que os aptâmeros, de fato, se ligam a Gal1, mas em um sítio afastado do CRD. Ensaios de hemaglutinação mostraram que os aptâmeros selecionados conseguiram inibir a ligação da Gal1 com as glicanas da superfície celular, bloqueando a atividade lectínica da proteína. Assim, esse conjunto de resultados mostram que foi possível o desenvolvimento de uma nova classe de inibidores da Gal1 baseada em aptâmeros de DNA, a partir de uma nova metodologia de SELEX, e que não atuam através dos mecanismos clássicos de bloqueio da atividade lectínica via CRD, abrindo nossas possibilidades no desenvolvimento de estratégias diagnósticas e terapêuticas envolvendo esta proteína. / Galectin-1 (Gal1) is a highly conserved lectin that recognizes ?-galactosides, involved in the regulation of cellular immunity tolerance and homeostasis. Data from the literature show that this endogenous lectin is widely expressed in sites of inflammation and tumorigenesis, directly participating in cell adhesion processes, tumor growth, metastasis and angiogenesis, highlighting the relevance of its detection in biological samples, and suggesting that its direct inhibition may result in benefits in the treatment of inflammatory disorders and in novel antitumor therapeutic strategies. However, data on Gal1 inhibitors with real therapeutic impact in blocking the biological activity of this lectin are still scarce. Aptamers are single-stranded oligonucleotides (DNA or RNA), which can bind to a wide variety of targets, such as ions, peptides, proteins, organic and inorganic molecules, with high affinity and specificity. The aptamers are selected from pools of random single-stranded oligonucleotide (ssDNA) sequences consisting of a variable central region, flanked by two sites of primers interaction for PCR amplification. This selection process is called Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this work, DNA aptamers that bind to recombinant human Gal1 and inhibit their lectin activity have been selected and characterized. The aptamers selection process was done through a variation of SELEX methodology, developed in this work and here called \"single vial selection\" (SVS), in which all stages of aptamers selection occurred in a single container, quickly and efficient, avoiding chromatographic steps, which are usually used in SELEX. Analyzes by Thermofluor (TSA) method and intrinsic tryptophan fluorescence spectroscopy have confirmed that aptamers actually bind to Gal1, but at a site away from the CRD. Hemagglutination assay showed that selected aptamers succeeded in inhibiting the Gal1 binding to cell surface glycans, blocking the protein lectin activity. Thus, this set of results showed that it was possible to develop a new class of Gal1 inhibitors based on DNA aptamers and on a new SELEX methodology, that does not act through the classic blocking mechanisms of lectin activity via CRD, opening new possibilities for the development of diagnostic and therapeutic strategies involving this protein.

La maladie d’Alzheimer est une pathologie neurodégénérative qui amène à une perte progressive de la mémoire et cause des changements comportementaux. Selon plusieurs théories, le développement de cette maladie est associé à l’accumulation du peptide amyloïde beta et de la protéine tau dans des zones précises du cerveau humain. A l’heure actuelle, les approches thérapeutiques testées sont fondées sur l’hypothèse de la cascade amyloïde, mais les résultats n’ont pas été jugés suffisamment efficaces. Pour augmenter les chances de succès des traitements thérapeutiques existants, de meilleures techniques pour un dépistage précoce de l’Alzheimer semblent nécessaires. De ce fait, dans cette thèse, des stratégies innovantes pour l’analyse d’un des biomarqueurs de la maladie d’Alzheimer sont proposées. En particulier le projet porte sur l’analyse de la protéine tau avec des biocapteurs basés sur la Résonance de Plasmons de Surface (SPR). L’augmentation du niveau de ce biomarqueur dans le Liquide Céphalo-Rachidien (LCR) est déjà indicateur d’un processus de neurodégénérescence. De plus, si la mesure de la protéine tau est combinée à celle d’autres biomarqueurs de la pathologie (i.e. : amyloïde beta), les possibilités de dépistage sont fortement augmentées. Les travaux ont portés sur deux aspects : initialement l’interaction antigène-anticorps a été exploitée pour développer un immunocapteur pour la protéine tau. En utilisant cette technologie, nous avons pu caractériser les paramètres analytiques de l’essai direct (avec un seul anticorps) et ceux de l’essai sandwich (avec deux anticorps complémentaires). Dès ces premières approches, nous avons remarqué le besoin d’augmenter la sensibilité de la méthode SPR développée. En effet la limite de détection pour l’essai sandwich était de l’ordre du nM, alors que les niveaux de tau dans le LCR sont de l’ordre du pM. L’utilisation de nanotechnologies, en particulier des nanotubes de carbone, a permis d’atteindre des niveaux proches du pM, avec de bonnes performances en terme de répétabilité de l’essai.Une approche alternative a été conçue dans la deuxième partie du projet. Elle était consacrée à la sélection d’un aptamère pour la protéine tau, afin d’exploiter les avantages de cette classe de récepteurs par rapport aux anticorps. Pour accomplir cet objectif, deux stratégies de sélection ont été mises en place. Premièrement la sélection traditionnelle (SELEX, Systematic Evolution of Ligands by EXponential enrichment) a été appliquée en utilisant l’Electrophorèse Capillaire (EC) comme moyen de séparation. Bien que de nombreuses conditions aient été modifiées, avec le SELEX traditionnel nous n’avons pas observé une évolution significative de l’affinité entre les séquences d’ADN et la protéine tau. Dans la deuxième approche nous avons utilisé la même méthode de séparation pour mener la sélection à travers l’EC-Non-SELEX. En utilisant cette méthode, où les étapes de PCR étaient réduites, une évolution positive a été observée après seulement trois rounds. En effet cinq séquences parmi celles issues du dernier round ont montré une affinité supérieure pour la cible par rapport à la banque. Néanmoins le nombre de séquences analysées à la fois par SPR et par anisotropie de fluorescence reste extrêmement limité par rapport au pool initial. Même si ceci semble être une limite, ce travail est le premier où les aptamères sont appliqués à l’analyse de la protéine tau. Le potentiel de cette classe de récepteurs reste en grande partie inexploré, ce qui laisse entrevoir des améliorations possibles de l’affinité grâce à de meilleurs processus de sélection et au développement de nouveaux outils bioinformatiques.En conclusion la SPR grâce à ses caractéristiques jouera un rôle fondamental dans les prochaines années pour l’analyse des biomarqueurs et pour le screening de nouvelles molécules, qui seront l’objet de futurs essais cliniques pour limiter l’agrégation de la protéine tau. / Alzheimer’s disease (AD) is a widespread pathogenic condition causing memory and behavior impairment mostly in elderlies because of the accumulation of amyloid beta peptide and tau protein in human brain. Current therapeutic approaches, based on the amyloid hypothesis, are unable to arrest the progression of the disease, hence early diagnosis is crucial for an effective intervention. Based on the updated criteria for AD probable diagnosis, and considering the limits associated with the actual analytical techniques, my work in this thesis was dedicated to develop novel strategies for AD diagnosis. The whole project focused on the analysis of tau protein by Surface Plasmon Resonance (SPR) biosensing. Such protein is well known for being relevant as neurodegenerative marker. In particular if the measurement of tau is associated with that of the amyloid beta peptide and that of the phosphorylated tau, the clinical specificity of this protein become significant to detect Alzheimer. Two aspects were studied; first of all an immunosensor was developed taking advantage of the well-established antigen-antibody interaction. After characterization of the analytical parameters of the direct assay (with primary antibody), a sandwich assay (using two monoclonal antibodies mapping on different analyte i.e. protein tau epitopes) was developed, allowing very low sensitivity to be obtained in artificial Cerebrospinal Fluid (aCSF). In particular to enhance the analytical signal Carbon Nano Tubes (CNTs) were used. Secondly, the research was focused on the selection of aptamers for tau. To this aim two SELEX (Systematic Evolution of Ligands by EXponential enrichment) methods were compared, both based on Capillary Electrophoresis (CE) for partitioning step of the process. Whether with CE-SELEX (first method), no significant affinity improvement was measured, using the CE-Non-SELEX (second method) affinity of the DNA library for tau protein was consistently improved. After isolation of a limited population of aptamer candidates, five sequences were chosen to be analyzed for their affinity for the target. Fluorescence Anisotropy (FA) measurements and SPR highlight similar behavior for the selected sequences, despite the detection principles of these techniques are significantly different. In conclusion the work highlight versatility of SPR technology used both for quantitative analysis and for new selected aptamers characterization in terms of affinity for the analyte tau. The above mentioned versatility is of great interest in a field such AD, which is rapidly expanding. Lowering the total tau levels has been recently identified as a new goal for therapy. Therefore many drug candidates are likely going to be tested in the near future. SPR technology is already widely used in pharmaceutical industry to investigate novel molecules, since it gives access to a large panel of information. In this panorama aptamer technology may improve the overall quality of the analytical data, allowing better comparison among drug candidates. With respect of these receptors, the thesis opened the door to new studies for DNA aptamers to recognize tau, with considerable advantages in term of the receptor stability. Moreover the whole potential of DNA aptamers selected in this work still remain to be explored. New selection methodologies, combined with fast progression of bioinformatics tools might give rise to affinity improvement, which will lead to sensitivity improvement for tau detection in the next few years.

Les aptamères sont des oligomères d'ADN ou d'ARN simple brin qui, en se repliant sous forme de structures tridimensionnelles peuvent avoir des interactions fortes et spécifiques envers un certain nombre de cibles. L'objectif de cette thèse a été de compléter les études existantes sur l'utilisation de l'électrophorèse capillaire (CE) et les aptamères afin de mettre au point une méthode de sélection d'aptamères par CE couplée à la fluorescence induite par laser et le séquençage haut-débit Illumina. Dans un premier temps, nous avons mis au point une méthode de détection et de séparation par électrophorèse capillaire couplée à la double détection UV-LEDIF d'une banque d'ADN en interaction avec une cible : la thrombine. C'est un modèle déjà étudié pour lequel deux aptamères ont fait l'objet de publications. Nous avons utilisé l'aptamère T29 dans le cadre de notre étude car c'est celui qui présente la meilleure affinité. L'électrophorèse capillaire est un puissant outil analytique qui facilite l'efficacité de sélection des aptamères et précise la détermination des paramètres d'interactions. Nous avons ainsi pu déterminer la constante d'affinité KD par CE-UV-LEDIF sur le modèle de base : la thrombine. Par ailleurs, nous montrons également comment l'utilisation du tampon Tris peut dégrader un ADN simple brin en électrophorèse capillaire et nous proposons comme alternative l'utilisation d'un tampon sodium phosphate dibasique qui évite ce phénomène de dégradation. Enfin, nous expliquons la difficulté d'amplification par qPCR et PCR d'un aptamère comme le T29 ayant une structure en G-quadruplex. Nous avons montré que le séquençage haut-débit Illumina nous a permis de trouver une corrélation entre le nombre de molécules séquencées et le nombre de séquences obtenues. L'analyse des séquences obtenues montre une quantité importante (20%) de séquences de T29 qui ne correspondent pas à la séquence de cet aptamère. Cela prouve que les étapes de PCR et de séquençage haut débit pour la détection de G-quadruplex peuvent induire un biais dans l'identification de ces molécules. / Aptamers are oligomers of small single-stranded DNA or RNA which can have strong and specific interactions with some targets when they fold into three-dimensional structures. The objective of this thesis was to complete existing studies on the use of capillary electrophoresis in order to develop a method for the selection of aptamers by CE coupled to laser induced fluorescence and Illumina high-throughput sequencing. In a first step, we developed a method of detection and separation by capillary electrophoresis coupled with the double detection UV-LEDIF of a DNA library interacting with a target: thrombin. It is a model already studied and for which two aptamers have been published. We used aptamer T29 as part of our study because it has the best affinity. Capillary Electrophoresis is a powerful analytical tool that facilitates the selection efficiency of aptamers and specifies the determination of the interaction parameters. We thus were able to determine the affinity constant KD by CE-UV-LEDIF on the basic model: thrombin. Moreover, we also show how the use of Tris buffer can degrade single-stranded DNA during capillary electrophoresis and we propose as an alternative the use of a dibasic sodium phosphate buffer which avoids the phenomenon of degradation. Finally, we explain the difficulty of amplification by qPCR and PCR of an aptamer such as T29 with a G-quadruplex structure. We showed that the Illumina high-throughput sequencing allowed us to find a correlation between the number of sequenced molecules and the number of sequences obtained. Analysis of the sequences obtained shows a significant amount (20%) of T29 sequences which do not correspond to the sequence of this aptamer. This shows that the PCR and high-throughput sequencing steps for the detection of G-quadruplex can induce bias in the identification of these molecules.

Thesis advisor: Jia Niu / Nucleic acid aptamers are promising alternatives to antibodies for a wide array of diagnostic and therapeutic applications. However, state-of-the-art aptamers suffer from poor pharmacokinetics and diversity, limiting their affinity and specificity for many therapeutically relevant targets. The emerging field of glycoscience provides opportunities to improve the utility of aptamers over antibodies. Combining synthetic chemistry with modern molecular biology and polymer science, the synthesis of Xeno Nucleic Acid monomers and chemoenzymatic polymerization via engineered polymerase enzymes allows the production of nucleic acid drugs with superior resistance to endogenous nucleases. The modular structure of nucleic acids provides for the design of sequence defined polymers capable of post-synthetically appending complex synthetic glycans, extending the catalytic geometry of aptamers. Our SELEX inspired FACS based particle display approach allows for high-throughput screening. Additionally, we expect this method has the capability of screening aptamers in human serum. Our synthetic approach utilizes a Sonogashira cross-coupling reaction to install a flexible alkyne to the major groove of 2′-deoxy-2′-fluoro-arabinose uracil base. By incorporated recent advances in nucleic acid synthesis, one-pot nucleobase activation and sugar glycosylation is achieved and bis-oxybenzyl phosphoamidite synthesis can afford gram scale HPLC-free purification of the triphosphates. The FANA C8-alkyl-uridine triphosphate will be incorporated by an engineered Tgo DNA polymerase to allow systematic introduction of alkynyl conjugation handles into a DNA-templated FANA polymer. Subsequent conjugation with azido-modified glycans via the Huisgen coppercatalyzed alkyne-azide cycloaddition (CuAAC) click reaction will generate sequence controlled nucleic acid-carbohydrate hybrid molecules amendable for directed evolution. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.

<p>Borane (BH<sub>3<sub>) chemistry offers unique chemical characteristics that enable boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate (BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs will interact in a novel way with the desired target molecules because they can coordinate with a diverse array of ligand sites in proteins or other RNA molecules. This is due to the distinct hydrophobicity, sterospecificity, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond. </p><p>The object of this dissertation is to explore the therapeutic applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate the immunogenicity of this modification. We used mouse cells to determine if BP-RNA would activate toll-like receptor (TLR 7), which is involved in innate immune response to foreign single stranded RNA (ssRNA). This response is undesired when applied to oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi, it would be an advantage to have low immunogenicity and high downregulation activity by the siRNA. To determine the innate immune activation of the BP-RNA through the TLR 7 we used a known activator, the human immunodeficiency virus (HIV) derived single-stranded RNA (ssRNA40) and measured the production of cytokines as a function of the number of modified BP-linkages. The production of cytokines IL-6 and TNF&#945; was quantified after the boranophosphate (BP), phosphorothioate (PS) or natural ssRNA40 were transfected into murine macrophage Raw264.7 cells. Natural and phosphorothioate RNA (PS-RNA) have been shown to be activators of TLR 7 receptors. In contrast, we found that fully modified BP- ssRNA40 did not activate TLR 7. This is relevant in oligonucleotide applications such as siRNA and RNA aptamers where off-target effects such as immune activation after administration are not desired. </p><p>Subsequently, the low immune activation would be an advantage when coupled to RNAi activity of the oligonucleotide. Thus, we explored whether BP modified siRNA molecules would modulate gene expression and if there was an effect on downregulation activity when increasing the number of BH3 modifications on the phosphate backbone. Our therapeutic model was the multi-drug resistance 1 (MDR1) gene that expresses P-glycoprotein (P-gp), which has been notoriously difficult to modulate. The aberrant regulation of genes such as MDR1 in cancer cells are a major cause of chemotherapeutic treatment failure against human cancers. Hence, controlling the expression of cancer genes with antisense technology is a possible cancer therapy. Specifically, correcting the overexpression of p-glycoprotein using modified siRNAs that target and degrade the P-glycoprotein mRNA produced by the MDR1 gene. We found that there is a reduction of siRNA activity with an increasing number of BP-modifications. It appears that there is a fine balance between lack of immune response and gene downregulation when applied to BP-siRNA. </p><p>Finally, we compared the enrichment during the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method of two libraries, one BP-RNA (U&#945;B) compared to a doubly-modified RNA (2'FC & U&#945;B), against a human thrombin. Aptamers modulate protein activity and interfere with protein signaling by binding to the desired protein with high affinity and specificity leading to their use in therapeutic applications where protein activity needs to be controlled or it is anomalous. In the case of blood coagulation, thrombin plays a central role in coagulation signaling cascade and it is a good target to use to control blood coagulation in clinical settings. We attempted to optimize the selection of BP- RNA aptamers through 4-8 rounds of SELEX against the protein thrombin. We found that the selection conditions were not optimal for BP-RNA SELEX possibly due to non-specific binding to a bovine serum albumin (BSA) in the selection buffer.</p> / Dissertation

Thrombosis is initiated by platelets and leads to cardio-, cerebro-, and peripheral vascular disease, the leading causes of morbidity and mortality in the western world. Antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use is limited by hemorrhage at high concentrations and sub-therapeutic activity at lower doses. Thus, development of new antiplatelet agents with improved safety and efficacy is a medical priority. VWF is a multimeric plasma glycoprotein that plays a critical role in platelet-mediated thrombus formation and presents an attractive target for antiplatelet therapy. To this end, I have isolated and characterized aptamer molecules that bind to VWF with high affinity and have shown that some of these aptamer molecules could inhibit platelet activation/aggregation in vitro and in vivo. Furthermore, I designed antidote molecules that can reverse the effects of the aptamer molecules, restoring platelet function quickly and effectively. This project has yielded the first antidote controlled antiplatelet agent and may lead to significant improvements in thrombosis therapy. Thrombin is a plasma protein that plays a critical role in thrombosis. Currently, available antithrombin agents are efficacious in preventing coagulation but do not significantly affect platelet activation and aggregation, both essential components of thrombus formation. Therefore, I tested two aptamer molecules that bind to mutually exclusive exosites on thrombin and, when used together, synergistically inhibit both coagulation and platelet activation. I demonstrated that this method could potentially lead to the development of effective antithrombotic therapies. With an ever-increasing number of people taking multiple medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs but unfortunately it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Therefore, I described the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents, i.e. aptamers, including those that I generated against VWF. I demonstrated that protein and polymer-based molecules that capture oligonucleotides can reverse the activity of aptamers in vitro and in vivo. / Dissertation

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.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 120-127).

abstract: A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences were isolated from a pool of random TNA molecules. This implicates a possible chemical framework capable of crosstalk between TNA and RNA. Further, this shows that heredity and evolution are not limited to the natural genetic system based on ribofuranosyl nucleic acids. Another alternative genetic system, glycerol nucleic acid (GNA) undergoes intrasystem pairing with superior thermalstability compared to that of DNA. Inspired by this property, I demonstrated a minimal nanostructure composed of both left- and right-handed mirro image GNA. This work suggested that GNA could be useful as promising orthogonal material in structural DNA nanotechnology. / Dissertation/Thesis / Ph.D. Chemistry 2011