Isolamento e caracterização de aptâmeros ligantes à 5’-UTR do genoma viral de SARS-CoV-2

Abstract

The development of technologies such as SELEX method for biomolecules investigation as those called aptamers has been applied for the purposes of theranostic and to produce new materials for organic and inorganic targets. Targeting the 5’-UTR region of the SARS-CoV-2 virus genome, ssDNA aptamers were selected in the present work. To obtain the sequence of SARS-CoV-2 as the target, the 5’-UTR of the virus genome was inserted in the pUC18 plasmid, for amplification, by symmetric and asymmetric PCR, and the production of ssDNA containing biotin complexed at its 5’-end. Similarly, the synthetic library of ligands was produced by symmetric and asymmetric PCR to obtain dsDNA and ssDNA, respectively, where the fragmenucleotídeos selected and purified presented a length of around 100 bp. Selection started with immobilizing the ssDNA 5’-UTRbio de SARS-CoV-2 on a magnetic platform, followed by incubation with the ssDNA library containing the potential oligonucleotides as ligands. After the removal of non-ligands, each round of selection was ended with symmetric and asymmetric PCR amplification of the ligands to the next round. Twelve selection rounds were performed, and the enrichment of the ligands to the target was evaluated by dot-blot, indicating affinity and specificity to the sequence of 5’-UTR of SARS-CoV- 2. Aptamers from the twelfth round were sequenced, presenting about 23 million raw data of sequences. Twelve ligands were analyzed in silico in a preliminary characterization, among which Apta01-5UTR-hCoV-2, Apta03-5UTR-hCoV-2, Apta03'-5UTR-hCoV-2, and Apta07-5UTR-hCoV-2 interacted with portions of SL 4.5 and SL 5 of the 5’-UTR of SARS-CoV-2. These results indicate that the aptamers anti-SARS-CoV-2 can be evaluated as theranostic tools from synthesis and/or chemical modifications of the oligonucleotides selected to perform new and complementary molecular and cellular tests.