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 (K D) 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.
Neomicrocalamus and Temochloa are closely related to bamboo genera. However, when considered with newly discovered and morphologically similar material from China and Vietnam, the phylogenetic relationship among these three groups was ambiguous in the analyses based on DNA regions. Here, as a means of investigating the relationships among the three bamboo groups and exploring potential sources of genomic conflicts, we present a phylogenomic examination based on the whole plastome, single-nucleotide polymorphism (SNP), and single-copy nuclear (SCN) gene datasets. Three different phylogenetic hypotheses were found. The inconsistency is attributed to the combination of incomplete lineage sorting and introgression. The origin of newly discovered bamboos is from introgressive hybridization between Temochloa liliana (which contributed 80.7% of the genome) and Neomicrocalamus prainii (19.3%), indicating that the newly discovered bamboos are closer to T. liliana in genetics. The more similar morphology and closer distribution elevation also imply a closer relationship between Temochloa and newly discovered bamboos.