DNA Aptamer (LepDapt) Against Lipl32 as A Potential Diagnostic Agent for Detection of Leptospira

Abstract

Leptospirosis is an infectious disease caused by pathogenic Leptospira spp. To enable
early detection, DNA aptamers (LepDapt) were developed to target LipL32, the most abundant outer membrane protein in pathogenic Leptospira. Among the identified candidates,LepDapt-5a exhibited the strongest binding affinity. Molecular dynamics (MD) simulations show that LepDapt-5a forms a stable G-quadruplex (G4) structure. While the G-quadruplex serves as the primary contributor to LepDapt-5a’s interaction with LipL32, the adjacent double helix enhances binding affinity by increasing the interaction surface. Analysis of perresidue binding energy via MM/PBSA highlights the significant roles of T19 and G24 in target interaction. To assess the impact of these residues, we used AlphaFold3 to predict the 3D structure of the DNA aptamers and docked them to LipL32 via HADDOCK2.4 webserver. MD simulations of all complexes were performed using the AMBER ff14SB and OL15 force fields for protein and nucleic acids, respectively. While the mutants preserved G4 formation, the G24 mutation disrupted the double helix structure but strengthened binding to LipL32. In contrast, mutation at T19 preserved the helical structure but weakened binding. The binding free energy (Δ𝐺) of LepDapt-5a, computed via the MM/PBSA method, was estimated to be –12.45 ± 12.99 kcal/mol. This value is consistent with experimental data of dissociation constants (Kd) of 33.97 ± 5.30 nM.