Ciencias Exactas y Ciencias de la Salud
Permanent URI for this collectionhttps://hdl.handle.net/11285/551039
Pertenecen a esta colección Tesis y Trabajos de grado de las Maestrías correspondientes a las Escuelas de Ingeniería y Ciencias así como a Medicina y Ciencias de la Salud.
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- Development of an aptazyme-driven toehold switch platform for colorimetric detection of SARS-CoV-2 N gene conserved regions(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2024) Esquivel Ortiz, Karla Mariel; Antonio Pérez, Aurora; emipsanchez; Benítez Cardoza, Claudia Guadalupe; School of Engineering and Sciences; Campus Estado de México; Torres Huerta, Ana LauraIn response to the urgent need for advanced diagnostic tools highlighted by the COVID-19 pandemic, this thesis project aimed to develop a novel, label-free colorimetric diagnostic platform using parallel G-quadruplex/hemin-aptazymes for detecting the SARS-CoV-2 virus. This work, rooted in designing these aptazymes in-silico that specifically target conserved regions of the SARS-CoV-2 N gene, namely TV1 and TV2, and to experimentally evaluate their potential as highly specific bioreceptors for biosensing technologies. First, extensive in-silico analyses were carried out to identify the potential targets and to evaluate the aptazyme sequence and interaction design. Multiple Sequence Alignment analyses helped us to identify SARS-CoV-2 N gene regions with high conservation across betacoronaviruses and SARS-CoV-2 variants present in Mexico, which would serve us as stable targets for our diagnostic platform. The designed aptazymes and identified target sequences were then studied for their secondary structure and stability at different working parameters using the mFold software. Tertiary structures were retrieved using RNAComposer, 3DNA, and PyMol software to evaluate structural changes at different working parameters. Finally, molecular docking simulations were carried out using PyDock DNA online server to predict the aptamer-target interactions. These initial studies determined the experimental working parameters in terms of ionic concentrations and provided an understanding of the molecules individually and in complex. Characterization techniques included Dynamic Light Scattering (DLS) to understand the size change behavior of the molecules, both individually and in complex; UV-Vis Spectroscopy to assess the aptazymes’ catalytic activity, analytical sensitivity and specificity, and capability of producing a colorimetric response when in complex; and Circular Dichroism (CD) to gain insights into the structural changes of the aptazymes alone and in complex. UV-Vis spectroscopy analyses revealed that aptazymes were non-specific to their targets, consistently demonstrating a colorimetric shift within the first five minutes of reaction. CD showed that the predominant structure observed after complex formation was the B-form hairpin topology for all aptazymes. These structural changes explained the non-specific results observed in UV-Vis spectroscopy. Consequently, the overallaptazyme sequence design should be refined. Following our aptazyme toehold design, increasing the length of the oehold sequence might improve the formation of a parallel G-quadruplex structure instead of a B-form hairpin. Further experiments or refinements are recommended to better characterize TV2 aptazyme which appeared to be a more promising andidate than TV1, based on its greater stability, fewer structural conformation, higher specificity, and lower limit of detection.