Simulated construction of tilmicosin nucleic acid aptamers based on molecular docking and molecular dynamics...

Traditional aptamer screening methods often prove ineffective for small molecule targets, primarily due to the inherent structural limitations of such compounds. Their simple architecture, limited functional groups, and restricted spatial complexity drastically reduce the probability of identifying nucleic acid sequences that bind with both high affinity and specificity. Consequently, the screening process becomes inefficient and labor-intensive, frequently failing to yield aptamers of satisfactory performance for practical applications. This represents a significant technical hurdle in expanding the use of aptamers in small-molecule detection and therapeutics.

Based on this, this study innovatively proposes an aptamer design method based on single-nucleotide docking assembly, using the small molecule temicloxacin as an example. Through molecular dynamics simulations (50 ns, RMSD convergence threshold of 0.15 nm), the dynamic conformational characteristics of tilmicosin were analyzed. Subsequently, saturated docking was performed on four classes of mononucleotides, screening out 32 high-affinity mononucleotides (atomic contact distance ≤4 Å). Methods such as depth-first search algorithm (DFS) and weighted graph theory model were introduced to obtain the representative single nucleotides of eight classes of functional modules and linkage assembly, and finally 63 non-redundant candidate sequences were screened. Molecular docking results indicate that the optimal aptamer Til-14 exhibits high binding affinity with tilmicosin. with an affinity of 298.16 ± 95.588 nM measured via SYBR Green I fluorescence assay. Colloidal gold colorimetric analysis confirmed its high affinity (Kd = 279.323 ± 87.234 nM) and excellent specificity.

This innovative method successfully addresses the key limitations of the traditional SELEX process in screening aptamers for small molecule targets. By enhancing the efficiency and specificity of selection, it not only facilitates the discovery of high-performance aptamers but also establishes a novel, generalizable framework for the construction of nucleic acid aptamers targeting other small molecules.