Investigação de alterações estruturais de aptâmeros ligantes à região 5’ não traduzida no genoma do vírus da dengue baseada em técnicas moleculares
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Data
2016-09-02
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Universidade Federal de Goiás
Resumo
The main purpose of this study was to assess conformational changes in preselected aptamers A03 and C10 against untranslated regions present in the genome of the dengue virus (DENV), by means of molecular biology and physics assays. Aptamers are nucleic acid molecules with potential theranostic applications. In this context, dengue was the target chosen for study because is an important infectious viral disease presenting high rates of morbidity and mortality in the worldwide, especially in tropical and subtropical areas, where there are high incidences of the mosquito vector Aedes aegypti. These aptamers showed, by dot-blot and magnetic beads ligand capture and subsequent qPCR detection, similar affinities and specificities to binding to the DENV 5'UTR. Probably, the analogy of the motifs and secondary structures presented in these ligands, identified by in silico studies, are responsible for these similarities. Data obtained by using Fluorescence Resonance Energy Transfer Assays (FRET) confirmed the affinity of A03 as DENV ligand by conformational changes in the double labeled aptamer (5’-FAM, 3’-TAMRA). Aptamer conformacional changes resulting from its interaction with the target were detected by the modifications in the fluorescence intensities between FAM and TAMRA fluorophores. These conformational changes results in donor-acceptor distance modification that has an effect on FRET efficiency and on fluorescence intensity of these molecules. The ionic strength of the buffer used in the trials had significant influence on the conformation of the aptamer, allowing the detection of interactions between A03 and DENV. The ions in the buffer lead an effect in the conformation of the aptamer, increasing its thermal stability, and the Mg2+ ion has led to major conformational changes on A03 compared to Na+ ion. The data presented herein demonstrated the importance of the studies of oligonucleotides binding affinity and specificity to Dengue virus. It can be concluded that the A03 and C10 aptamers have potential in application for diagnostic and/or therapeutic purposes, considering the diluent buffer, since different salts have influence on the secondary and tertiary conformational structures of aptamers, and on bioavailability of interaction with the target.
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Aptâmeros, DENV, Dot blot, PCR em tempo real, FRET, Aptamer, Real-time PCR
Citação
ARRUDA, R. A. R. Investigação de alterações estruturais de aptâmeros ligantes à região 5’ não traduzida no genoma do vírus da dengue baseada em técnicas moleculares. 2016. 81 f. Dissertação (Mestrado em Química) - Universidade Federal de Goiás, Catalão, 2016.