Computational design and experimental evaluation of a biotinylated DNA aptamer against Entamoeba histolytica actin

Amoebiasis, caused by Entamoeba histolytica, remains a global health concern due to its asymptomatic nature and potential for severe complications such as liver abscesses. To investigate molecular recognition mechanisms, this study focused on the binding affinity and detection limit of aptamers targeting E. histolytica actin (EhActin), a cytoskeletal protein selected as a target molecule. A genomic tRNA-based sequence library was computationally screened to identify potential high-affinity aptamer candidates, followed by molecular docking and molecular dynamics simulations to evaluate interaction profiles. Recombinant EhActin (rEhActin) was expressed in Escherichia coli LEMO21 using a pET28a(+) vector system and used in ELONA to quantify the interaction between a biotinylated DNA aptamer and rEhActin. The limit of detection was also assessed using ELONA with E. histolytica protein extract. Among the shortlisted aptamers, APT29 demonstrated favorable in silico and in vitro binding characteristics. Molecular dynamics analysis supported overall structural stability of the APT29-EhActin complex, although hydrogen bond interactions fluctuated over time. ELONA showed a dissociation constant (Kd) of 44.72 nM for APT29, and this aptamer achieved a limit of detection of 0.4 µg/well of E. histolytica protein extract. These findings suggest APT29 as a promising molecular probe for further biophysical and structural studies of aptamer-target interactions involving E. histolytica, with potential relevance to future diagnostic development for amoebiasis.