Selection and validation of novel DNA aptamer panel co-specific to Plasmodium falciparum and Plasmodium ovale lactate dehydrogenase.

Malaria remains a major global health concern. The development of point-of-care diagnostic aptasensors could improve malaria detection, patient management, and eradication efforts. We selected and validated single-stranded DNA aptamers against Plasmodium falciparum lactate dehydrogenase (Pf-LDH), using systematic evolution of ligands by exponential enrichment (SELEX). The five most abundant aptamers were validated with an enzyme-linked oligonucleotide assay (ELONA). The aptamer binding affinities to Pf-LDH varied between 0.35 and 3.08 nM. Truncation of aptamers enhanced binding affinities in most cases, with all truncated aptamers showing comparable or improved dissociation constants (Kd) relative to full-length aptamers, except Pf-LDH5t, which lost its binding affinity. Selectivity analysis against LDH from P. ovale, P. vivax, and P. malariae showed that Pf-LDH1 and Pf-LDH1t specifically recognized Pf-LDH (Kd = 2.74 ± 0.28 and 1.13 ± 0.26 nM, respectively), while Pf-LDH2, Pf-LDH3, Pf-LDH4, and their truncated forms, as well as Pf-LDH5, recognized both Pf-LDH and P. ovale LDH (Po-LDH). Crystallography of Pf-LDH4t revealed a binding site overlapping that of the previously characterized aptamer 2008 but involving distinct molecular interactions. These findings demonstrate the structural versatility of Plasmodium LDH-specific aptamers and provide a novel Pf- and Po-LDH co-specific aptamer panel with strong potential for next-generation aptamer-based malaria diagnostics.