Functional nucleic acids (FNAs), which include both DNA/RNA aptamers and DNA enzymes, have emerged as promising biological recognition elements for biosensors. These species typically require immobilization on or within a solid support, which is usually interfaced to some kind of signal transducer and readout system when use in biosensor. Our group has successfully immobilized several functional nucleic acids in the past, including fluorescence-signalling DNA enzymes, DNA aptamers and RNA aptamers by entrapping them into porous silica or organosilica materials prepared by the sol-gel method using percursors such as sodium silicate (SS), diglyceryl silane (DGS), tetrametylorthosilicate (TMOS) and trimethoxymethysilane (MTMS). While the earlier work established the ability of entrapped FNAs to retain binding and catalytic activity, only limited information was obtained on how different factors affect the performance of immobilized FNAs, and no information was obtained on the effects of aging and storage conditions on FNA performance. The initial objective of this thesis was to employ advanced fluorescence methods to better understand the nature of immobilized DNA and RNA aptamers, and in particular how entrapment in different sol-gel based materials affected FNA performance for detection of small molecule analytes. It was found that the ability of the entrapped aptamer reporters to remain fully hybridized was the most important factor in terms of signalling capability for both DNA and RNA aptamer reporters. It was also observed that more polar materials derived from SS were optimal for both types of aptamer reporters, since these allowed the entrapped aptamers to remain hydridized to their complementary strands and still retain the dynamic motion needed to undergo structure switching, while providing a minimum degree of leaching. The second objective of my research was to develop a paper-based biosensing device incorporating immobilized DNA and RNA aptamers that could be used in the fields of point-of-care diagnostics to further expand the utility of structure-switching aptamer reporters to real world application. A dual response (fluorescence / colorimetric) paper-based sensor utilized printed graphene oxide to immobilize both a RNA and a DNA aptamer in a recognition zone. Upon target addition, the aptamer desorbed and eluted to an amplification zone where rolling circle amplification was used to generate a colorimetric output. This sensor could function with clinical samples such as serum and stool, and allowed detection of key bacterial markers (ATP and glutamate dehydrogenase) at clinically relevant levels. / Thesis / Doctor of Philosophy (PhD)

Les ARN (Acide RiboNucléique) remplissent de nombreuses fonctions clés dans le vivant. Ils peuvent être support de l'information génétique, régulateurs de celle-ci. Visualiser ces molécules au sein d'une cellule représenterait une étape importante vers une meilleure compréhension de la régulation de l'expression des gènes. Les ARN fluorogènes tels que Spinach et Mango sont des outils extrêmement prometteurs pour atteindre cet objectif. Cependant ces deux ARN fluorogènes présentent une brillance limitée. La Compartimentation in vitro assistée par microfluidique (µCIV) est un outil très prometteur dont notre groupe a démontré l’efficacité pour l’évolution d’ARN. Dans le cadre de cette thèse, la µCIV a été adaptée à la sélection d'aptamères d'ARN fluorogènes pour en améliorer les propriétés (surtout la brillance). De plus, l’utilisation conjointe du séquençage haut débit a permis l’optimisation très rapide et semi-automatisée à la fois d’aptamères mais aussi de biosenseurs fluorogènes. Ainsi, cette thèse a permis de mettre en place et d’exploiter des technologies de criblage robustes pour la découverte de nouveaux aptamères d'ARN et de biosenseurs. / RNA is a key molecule in gene expression and its regulation. Therefore, being able to monitor RNA through live-cell imaging would represent an important step toward a better understanding of gene expression regulation. RNA-based fluorogenic modules are extremely promising tools to reach this goal. To this end, two light-up RNA aptamers (Spinach and Mango) display attractive properties but they suffer from a limited brightness. Since previous work in the group demonstrated the possibility to evolve RNA using microfluidic-assisted in vitro compartmentalization (µIVC), this technology appeared to be well suited to improve light-up aptamers properties by an evolution strategy. Therefore, the µIVC procedure was adapted to fluorogenic RNA aptamers to improve their properties (especially the brightness). Finally, using µIVC in tandem with high-throughput sequencing (NGS) allowed further developing the technology into a more integrated and semi-automatized approach in which RNAs and biosensors are selected by µIVC screening and the best variants identified by a bioinformatics process upon NGS analysis. To summarize, this thesis allowed establishing robust µIVC screening workflows for the discovery of novel efficient light-up RNA aptamers as well as metabolites biosensors.

The work described here details the experimental progress toward an improved means of HIV-1 diagnosis and an explanation of the experimental approaches taken to advance a previously developed HIV-1 reverse transcriptase detection assay using RNA aptamers for protein capture. After characterization of the identity and function of the aptamer samples to be used, we first set about clarifying the nature of the assay and pinning down sources of variability inherent in the original Aptamer Antibody Sandwich Assay (AASA) such that through the course of this work we might bring the assay to a point of high reproducibility. In doing so, we devised a set of criteria for data analysis and filtration and established a process to examine whether modifications to the method resulted in measurable improvement. Two new methods were tested in the hope that they might later be extended to our ultimate project goal of distinguishing binding affinity variations among HIV-1 reverse transcriptase protein and its mutant variants. Both method modifications involved the addition of a fluorescently labeled Cy5 probe to the immobilized aptamer construct. The addition of a fluorescent label to each printed aptamer allowed for detection of aptamer presence in addition to protein binding, essentially serving as a simple internal control for aptamer-protein binding. After optimizing the AASA aptamer construct and experimental procedure, the AASA was extended to a multiplexed array format. Using four groups of aptamers selected against two HIV-1 RT variants (wild-type and mutant 3) we tested the hypothesis that immobilized anti-HIV-1 aptamers might be capable of binding HIV-1 RT variants and regardless of their selective target. The experiments described here are the first example of these aptamers being used in a multiplexed array format, and the results are not only a clear exemplification of the capacity of RNA aptamers for detection in this novel, immobilized assay format, but also an indicator of the utility and flexibility of RNA aptamer functionality. The promising results described in these preliminary studies are the starting block from which several interesting aptamer-protein interaction and drug-competition studies have begun. / text