Rational design and molecular dynamics study of LipL32-targeting DNA aptamers for diagnostic applications

Leptospirosis is an infectious disease caused by pathogenic Leptospira spp., to mitigate severe outcomes, early detection is needed. DNA aptamers (LepDapt) targeting LipL32, the most abundant outer membrane protein in pathogenic Leptospira, were developed to enable early detection. Among the identified candidates, LepDapt-5a exhibited the strongest binding affinity, attributed to its stable G-quadruplex (G4) structure, enhanced by adjacent double-helix regions that increase the interaction surface with LipL32. Molecular dynamics (MD) simulations, using AMBER ff14SB and OL15 force fields for protein and nucleic acids, respectively, show that LepDapt-5 aptamers family forms a stable G-quadruplex (G4) structure. Analysis of per-residue binding energy via MM/PBSA of LepDapt-5a…LipL32 complex highlights the significant roles of G7, T19, and G24 in target interaction. To assess the impact of these residues, we introduced single-point mutation at these positions to their complementary base pair and denoted them as G7C, T19A, and G24C, respectively. AlphaFold3-predicted 3D structure of these aptamers were then docked to LipL32 via HADDOCK2.4 webserver, followed by MD simulations. While the mutants preserved G-quadruplex structure integrity, they altered the binding mechanism of LepDapt-5a. G7C shifted the dominant interaction from G-quadruplex to the adjacent double helix, G24 mutation enhanced binding by disrupting the double helix structure into head and tail subunits, while T19A weakened affinity despite maintaining helical structure. The binding free energy (ΔG) of LepDapt-5a, computed via the MM/PBSA method, was estimated to be -14.99 ± 12.80 kcal/mol. This value is consistent with experimental data of dissociation constants (Kd) of 33.97 ± 5.30 nM. ELASA experiments confirmed the binding trend: G24C > WT ≈ G7C > T19A, validating the non-equilibrium alchemical predictions. These findings elucidate the structural basis of aptamer-LipL32 interactions, highlighting the role of not just G-quadruplex but also the adjacent units in optimizing binding affinity. This work informs the design of next-generation aptamers for rapid and specific Leptospira detection, addressing critical diagnostic challenges.