Ciencias Exactas 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 Laura
In 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.
Application and evaluation of aptamers in the design of the biosensor for the capture of testosterone and/or its synthetic analogs in food supplements.
(Instituto Tecnológico y de Estudios Superiores de Monterrey, 2024) Medina Benítez, Ariadna; Antonio Pérez, Aurora; emimmayorquin; Aranda Barrandas, Maria Eugenia; School of Engineering and Sciences; Campus Estado de México; Torres Huerta, Ana Laura
The present project arises from the need to develop a detecting tool for the presence of testosterone and its synthetic analogs in food supplements. Testosterone and its synthetic analogs are part of the androgenic-anabolic steroids (AASs) that are also cataloged as prohibited substances for food supplements since they have several adverse effects. The presence of AASs poses a significant public health issue due to several factors. These include the lack of strict regulations, the rise in consumption of food supplements, and the challenges associated with current analytical methods for assessing supplement composition. In response to this growing problem, the project searches to develop a competitive detection tool with high affinity, real-time detection, and user-friendly operation. To achieve this, the development of a biosensor was proposed, focusing on its two main components: (1) the biorecognition element, utilizing aptamers for their ability to specifically recognize and capture small analytes, and (2) the transducer element, employing gold nanoparticles (AuNPs) for their chemical and optical properties, which enable them to attach to aptamers via thiol modification. For this study, eight aptamers (apT5, P4G13, TESS1, TESS2, TESS3, T4, T5.1, and T6) with previous reports of interaction with testosterone were evaluated to select the best candidate regarding the structural stability at different ionic concentrations and temperatures values, as well as best affinity and interaction characteristics with testosterone, androstenedione, and testosterone undecanoate. In silico analysis allows us to know the stability and behavior of each analyzed aptamer and evaluate the aptamer’s ability to interact with its target. On the other hand, an in vitro analysis tracks the changes in the folded and unfolded aptamer dimensions monitored by the Dynamic Light Scattering (DLS) technique and the structural changes using the Circular Dichroism (CD) technique, both under the previously in silico conditions. An intermediate step was to construct and characterize the size of the biosensing platform (aptamer-AuNPs) and the binding complex (aptamer-AuNPs-analyte) using the DLS technique. To analyze the affinity with the analytes we performed relative capture tests with testosterone and testosterone undecanoate. The results from the in silico approach were consistent with the experimental data gathered, exhibiting that the sequence size in bp, and aptamers native conformation, intervene in their affinity and biosensor functionality. The aptamers apT5, P4G13, and T4 were discarded as possible candidates because they did not show stability and optimal affinity in the in silico analysis. T5.1, and T6 proved to be functional aptamers because they showed good affinity but less stability, both in silico and in vitro, TESS2, and TESS3, showed optimal stability and affinity, however, TESS1 turned out to be the best candidates because of their stability and number of interactions, which translates into a greater affinity, compared to the rest.

Ciencias Exactas y Ciencias de la Salud

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