Structure-Guided Optimization of a LipL32-Targeting DNA Aptamer through Molecular Dynamics and Free-Energy Analysis
4 affiliations
- 1Thammasat University , , , ,
- 2Kanazawa University , , ,
- 3Universiti Sains Malaysia , , Bertam , , ,
- 4MILA University , , No 1, MIU Boulevard, Putra Nilai , , ,
Abstract
Accurate detection of leptospirosis requires high-affinity recognition of the conserved LipL32 biomarker. Although LipL32-targeting DNA aptamers have been identified through systematic evolution of ligands by exponential enrichment (SELEX), the molecular basis of their target recognition remains unclear, limiting rational post-SELEX optimization. Here, we investigated the molecular recognition mechanism of the LepDapt-5 aptamer family using molecular dynamics (MD) simulations and binding free-energy analysis. We generated three-dimensional aptamer models using AlphaFold 3 (AF3) and constructed protein–aptamer docking complexes with HADDOCK, showing a stable G-quadruplex (GQ) motif within the aptamer core. Structural and energetic analyses identified key domains contributing to LipL32 binding, while per-residue energy decomposition highlighted nucleotides suitable for rational sequence refinement. Guided by these insights, we introduced targeted single-point mutations, evaluated their effects computationally, and validated their performance experimentally using enzyme-linked aptamer sorbent assays (ELASA). Relative binding free energies (ΔΔGbind) calculated using nonequilibrium alchemical MD showed strong agreement with ELASA measurements: T19A reduced binding affinity, G7C produced minimal change, and G24C significantly enhanced affinity. Experimental equilibrium dissociation constants (KD) further supported these predictions, identifying G24C as the highest-affinity variant. Additionally, the G24C mutant retained strong binding to LipL32 while exhibiting no detectable binding to the NS1 protein of dengue virus 2 and the E2 protein of Chikungunya virus.
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BibTeX
@article{vu2026structureguided,
title = {{Structure-Guided Optimization of a LipL32-Targeting DNA Aptamer through Molecular Dynamics and Free-Energy Analysis}},
author = {Tri Cao Vu and Boonchoy Soontornworajit and Pichayanoot Rotkrua and Jiraporn Arunpanichlert and Cong Quang Vu and Marimuthu Citartan and Yuthana Tantirungrotechai},
journal = {ACS Omega},
year = {2026},
month = oct,
doi = {10.1021/acsomega.6c03816},
url = {https://pubs.acs.org/acsodf/article/11/42/64524/5452283/Structure-Guided-Optimization-of-a-LipL32},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Structure-Guided Optimization of a LipL32-Targeting DNA Aptamer through Molecular Dynamics and Free-Energy Analysis
AU - Tri Cao Vu
AU - Boonchoy Soontornworajit
AU - Pichayanoot Rotkrua
AU - Jiraporn Arunpanichlert
AU - Cong Quang Vu
AU - Marimuthu Citartan
AU - Yuthana Tantirungrotechai
PY - 2026
DA - 2026/10/15
JO - ACS Omega
DO - 10.1021/acsomega.6c03816
UR - https://pubs.acs.org/acsodf/article/11/42/64524/5452283/Structure-Guided-Optimization-of-a-LipL32
AB - Accurate detection of leptospirosis requires high-affinity recognition of the conserved LipL32 biomarker. Although LipL32-targeting DNA aptamers have been identified through systematic evolution of ligands by exponential enrichment (SELEX), the molecular basis of their target recognition remains unclear, limiting rational post-SELEX optimization. Here, we investigated the molecular recognition mechanism of the LepDapt-5 aptamer family using molecular dynamics (MD) simulations and binding free-energy analysis. We generated three-dimensional aptamer models using AlphaFold 3 (AF3) and constructed protein–aptamer docking complexes with HADDOCK, showing a stable G-quadruplex (GQ) motif within the aptamer core. Structural and energetic analyses identified key domains contributing to LipL32 binding, while per-residue energy decomposition highlighted nucleotides suitable for rational sequence refinement. Guided by these insights, we introduced targeted single-point mutations, evaluated their effects computationally, and validated their performance experimentally using enzyme-linked aptamer sorbent assays (ELASA). Relative binding free energies (ΔΔGbind) calculated using nonequilibrium alchemical MD showed strong agreement with ELASA measurements: T19A reduced binding affinity, G7C produced minimal change, and G24C significantly enhanced affinity. Experimental equilibrium dissociation constants (KD) further supported these predictions, identifying G24C as the highest-affinity variant. Additionally, the G24C mutant retained strong binding to LipL32 while exhibiting no detectable binding to the NS1 protein of dengue virus 2 and the E2 protein of Chikungunya virus.
SN - 2470-1343
ER -