<i>In\nSilico</i> Analysis of Binding Sites\nfor a Novel ssDNA Aptamer Specific to Verrucarin A and Detection in\nDust Extracts

An aptamer is a single-stranded oligonucleotide\nthat\nserves as\na chemical antibody with a high specificity and binding affinity that\ncan recognize a wide range of molecules. Effective modification and\ntruncation of aptamers can enhance their binding affinities to particular\ntargets while also broadening their application for uses, such as\nbiosensors. However, a conventional trial-and-error methodology hinders\nthis process. Herein, we demonstrate an <i>in silico</i> method to elucidate the binding site of single-stranded DNA aptamer\nspecific to verrucarin A, a mycotoxin produced by molds in indoor\nbuildings that causes adverse effects in living organisms. The novel\nssDNA aptamer exhibited a binding affinity of 29.5 nM, demonstrating\na relatively strong affinity compared to those of previously reported\ntypical aptamers for small molecules. Furthermore, the selected aptamer\nwas highly specific toward verrucarin A among structurally related\nmycotoxins (i.e., verrucarol and zearalenone). The specific binding\nsite of the aptamer predicted via molecular dynamics and molecular\ndocking simulations was highly consistent with the results observed\nvia truncation, single base mutation, and circular dichroism experiments.\nThe fluorescence assay revealed limits of detection and quantification\nof 4.1 and 12 nM for the aptamer, respectively. Comparing our developed\naptasensor with LC–MS/MS methodology revealed that it could\ndetect verrucarin A levels in phosphate-buffered saline and dust extracts\nwith robust precision and consistency. Our findings provide insight\nfor future studies exploring interaction mechanisms with intended\ntargets and practical sensing applications, such as point-of-care\ndetection of verrucarin A.