Resumen de Obtención de aptámeros específicos para enzimas de la vía del metileritritol fosfato de microorganismos
Background: Pathogenic microorganisms such as the bacteria Mycobacterium tuberculosis and Pseudomonas aeruginosa, and the protozoa of the phylum Apicomplexa, including the causing agents of malaria and toxoplasmosis, synthesize the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), by the methylerythritol phosphate (MEP) pathway. This pathway is essential for most bacteria and Apicomplexa, but it is not present in humans, which synthesize IPP and DMAPP by the alternative mevalonate pathway. The essential role of the MEP pathway and its distribution in different organisms make their enzymes attractive targets for the development of new anti-infective agents. Herein, we focus on the development of aptamers against key enzymes of the MEP pathway. Aptamers are single-stranded oligonucleotides which behave as “chemical antibodies” and can bind specifically and efficiently to a given target molecule. Methods: Several methods have been optimized, such as: (i) the production of MEP pathway enzymes, (ii) the development of aptamers through systematic evolution of ligands by exponential enrichment, an in vitro selection process based on iterative cycles of binding, partitioning, and amplification of oligonucleotides from a pool of variant sequences, (iii) the cloning of aptamers, (iv) the establishment of an electrophoretic motility shift assay for the identification of interactions between selected aptamers and their target enzymes, and (v) methods for the in vitro evaluation of enzymatic activity. Results: We report the identification of a DNA aptamer (D10) which specifically binds to the enzyme catalyzing the first committed step of the MEP pathway: 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). Conclusions: The results obtained suggest that the D10 DNA aptamer could be a potential candidate for the development of new therapeutic agents and for the design of novel diagnosis systems.
