Development of DNA Aptamers Against <i>Plasmodium falciparum</i> Blood Stages Using Cell-Systematic Evolution of Ligands by EXponential Enrichment.

Abstract

"New biomarkers have to be developed in order to increase the performance of current antigen-based malaria rapid diagnosis. Antibody production often involves the use of laboratory animals and is time-consuming and costly, especially when the target is " - ", whose variable antigen expression complicates the development of long-lived biomarkers. To circumvent these obstacles, we have applied the Systematic Evolution of Ligands by EXponential enrichment method to the rapid identification of DNA aptamers against " - "-infected red blood cells (pRBCs). Five 70 b-long ssDNA sequences, and their shorter forms without the flanking PCR primer-binding regions, have been identified having a highly specific binding of pRBCs versus non-infected erythrocytes. Structural analysis revealed G-enriched sequences compatible with the formation of G-quadruplexes. The selected aptamers recognized intracellular epitopes with apparent " - "s in the " - "M range in both fixed and non-fixed saponin-permeabilized pRBCs, improving >30-fold the pRBC detection in comparison with aptamers raised against " - " lactate dehydrogenase, the gold standard antigen for current malaria diagnostic tests. In thin blood smears of clinical samples the aptamers reported in this work specifically bound all " - " stages versus non-infected erythrocytes, and also detected early and late stages of the human malaria parasites " - ", " - " and " - . The results are discussed in the context of their potential application in future malaria diagnostic devices.