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| Author(s): |
Rafael Augusto Lopes Franco
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Araraquara. 0000-00-00. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara |
| Defense date: | 2021-06-28 |
| Advisor: | Danielle Biscaro Pedrolli |
| Abstract | |
Dengue and Zika are arboviruses of the Flavivirus genus and represent a risk of infection for millions of people worldwide. In Brazil, 2019, the number of reports for these diseases increased by 650% for Dengue and 65% for Zika compared to the previous year. Even with the high incidence the number of precise confirmed cases (by serology or molecular detection) is low. Currently, the precise diagnosis of these arboviruses is made by techniques such as ELISA and RT-PCR, that require particular laboratory resources and the handling by technicians, which makes the diagnosis expensive and time-consuming. In addition, the demanded laboratory structure makes the diagnosis impracticable in remote regions of Brazil and also in other affected tropical countries. This work aimed to develop a synthetic DNA circuit for the rapid and simple identification of viral RNA from Zika and Dengue in an in vitro gene expression system. Toehold Switch previously developed in the SynBio AQA research group (called switch DENV10550(2) and switch ZIKV1440(2.6)) capable of detecting, individually, the viral RNA (trigger RNA) to the two diseases were employed. These riboregulators were used in 3 different signal amplification strategies: one based on the Mf-Lon protease, another based on the T7 RNA polymerase enzyme, and the third in a system using serine-type integrase. In vitro gene expression reactions are performed to evaluate the response of the synthetic circuits in the presence of specific and non-specific trigger RNA. The activation of the T7 RNApol systems was measured by the activity of reporter protein luciferase. In the evaluation of the Mf-Lon protease circuit, the green fluorescent protein fused to the degradation tail pdt#5 was used to assess the response. The Mf-Lon protease system in vitro gene expression assays did not indicate high degradation rates of the reporter protein, the fluorescence loss observed was, mainly, due to GFP quenching after repeated excitation cycles. For the signal amplification system based on serine-type integrase, due to several adversities, it was not possible to build the response unit. Only the detection plasmids with the integrase Int13 and Int9 were constructed. These contain the DENV10550(2) switch controlling the integrase gene The highest fold change observed in the T7 RNApol system was with the circuit separated in two plasmids and employing the DENV10550(2) switch, a response 2.29 times greater was achieved in the presence of 500 ng of RNA specific trigger (trigger DENV10550) in comparison to the response with the non-specific RNA. The T7 RNApol system with the ZIKV1440(2.6) showed a smaller fold change to the specific RNA trigger (trigger ZIKV1440) in relation to the response with the non-specific RNA trigger (trigger DENV10550), due to this fact the switches used in the work were re-evaluated, and it was found that the ZIKV1440(2.6) switch is not functional. The response of the T7 RNApol device with the DENV10550(2) switch shows the functionality and specificity of this signaling strategy, however, the fold change achieved was slightly higher than that obtained in the device with a direct response circuit. (AU) | |
| FAPESP's process: | 18/24272-3 - Construction of a signal amplifier system for riboregulators |
| Grantee: | Rafael Augusto Lopes Franco |
| Support Opportunities: | Scholarships in Brazil - Master |