Isolamento de aptâmeros ligantes à sequência 3'-UTR do RNA do vírus da dengue
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Data
2015-09-09
Autores
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Universidade Federal de Goiás
Resumo
Considering potential molecular methods for diagnostic and/or therapeutic
purpose of infectious human diseases, such as dengue, highlights the
systematic evolution of ligands by exponential enrichment, known as SELEX. In
this method, ligands to a target are exponentially enriched generating aptamers
of DNA or RNA with high specificity, being considered as artificial antibodies. In
this work, we aimed to realize the isolation of aptamers binding to 3'-UTR
(untranslated region) of the RNA from dengue virus (DENV), because present
important conformational structures that act as functional elements into RNA
necessary in the infectious process. Total RNA of C6/36 infected cells was
extracted, submitted to reverse transcription reaction to obtain viral cDNA
(serotypes 2 and 3) and amplified by symmetric and/or asymmetric PCR
technique to produce the 3’-UTR from DENV as target, generating RNA-like by
containing deoxyuridine triphosphate (dUTP) and biotin in the 5’ region of the
target. The random library of RNA ligands was obtained by sonication of a pool
from human genomic DNA used in three successive PCR, and in the last
reaction was introduced the promoter of T7 RNA polymerase, presenting
fragments ranging from 80 to 600-bp. Eight rounds of selection were performed
between the target and the library by using paramagnetic particles coated with
streptavidin. For each round, after the incubation, the non-ligands were
removed by using magnetic platform, and the ligands were eluted with NaOH.
The eluted ligands were precipitated, submitted to RT-PCR and transcription in
vitro, completing one round of selection. For analysis of variability of ligands, the
product obtained from the eighth round was cloned and fourteen clones were
randomly selected for amplification. The results demonstrated that the aptamers
presented sizes with estimated molecular weights varying from 80 to 100-bp.
These data indicated the viability of aptamers isolation against conformational
elements present in the 3'-UTR of RNA from dengue virus, which may
contribute to future research focusing on prevention and/or control of the
disease.
Descrição
Palavras-chave
SELEX, Ácido ribonucleico, Aedes aegypti, DENV, SELEX, Ribonucleic acid, Aedes aegypti, DENV
Citação
SILVA, A. G. Isolamento de aptâmeros ligantes à sequência 3'-UTR do RNA do vírus da dengue. 2015. 65 f. Dissertação (Mestrado em Química) - Universidade Federal de Goiás, Goiânia, 2015.