RESUMEN
Dysregulation of tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, causes several diseases, making it an important therapeutic target. Here, we identified a novel DNA aptamer against human TNFα using inâ vitro selection, which included a high exclusion pressure process against non-binding and weak binders through microbead-assisted capillary electrophoresis (MACE) in only three rounds. Among the 15 most enriched aptamers, Apt14 exhibited the highest inhibitory activity for the interaction between TNFα and its cognate receptor in mouse L929 cells. For further improving the bioactivity of the aptamer, dimerization programed by hybridization was evaluated, resulting in the Apt14 dimer exhibited a twofold higher binding affinity and stronger inhibition compared to the monomer counterpart. Rapid identification of bioactive aptamers using MACE in combination with facile dimerization by hybridization accelerates the discovery of novel bioactive aptamers, paving the way toward replacing current monoclonal antibody therapy with the less expensive and non-immunogenic aptamer therapy.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Descubrimiento de Drogas , Técnica SELEX de Producción de Aptámeros , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Aptámeros de Nucleótidos/síntesis química , Aptámeros de Nucleótidos/química , Línea Celular , Electroforesis Capilar , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Vascular endothelial growth factors (VEGFs) are hypoxia-inducible secreted proteins to promote angiogenesis, in which VEGF-A is an important molecule that binds and activates VEGF receptor-1 (VEGFR-1) and VEGFR-2. In this study, two DNA aptamers, Apt01 and Apt02, were successfully isolated by alternating consecutive systematic evolution of ligands by exponential enrichment (SELEX) against VEGFR-1 and -2 using deep sequencing analysis in an early selection round. Their binding affinities for VEGFR-2 were lower than that of VEGFR-1, which is similar to that of VEGF-A. Structural analyses with the measurements of circular dichroism spectra and ultraviolet melting curve showed that Apt01 possessed the stem-loop structure in the molecule, whereas Apt02 formed G-quadruplex structures. In addition, Apt02 accelerated a tube formation of human umbilical vein endothelial cells faster than Apt01, which was affected by difference of binding affinity and nuclease resistance due to G-quadruplex structures. These results demonstrated that Apt02 might have a potential to function as an alternative to VEGF-A.