In Silico Analysis of Binding Sites for a Novel ssDNA Aptamer Specific to Verrucarin A and Detection in Dust Extracts

Abstract

An aptamer is a single-stranded oligonucleotide that serves as a chemical antibody with a high specificity and binding affinity that can recognize a wide range of molecules. Effective modification and truncation of aptamers can enhance their binding affinities to particular targets while also broadening their application for uses, such as biosensors. However, a conventional trial-and-error methodology hinders this process. Herein, we demonstrate an in silico method to elucidate the binding site of single-stranded DNA aptamer specific to verrucarin A, a mycotoxin produced by molds in indoor buildings that causes adverse effects in living organisms. The novel ssDNA aptamer exhibited a binding affinity of 29.5 nM, demonstrating a relatively strong affinity compared to those of previously reported typical aptamers for small molecules. Furthermore, the selected aptamer was highly specific toward verrucarin A among structurally related mycotoxins (i.e., verrucarol and zearalenone). The specific binding site of the aptamer predicted via molecular dynamics and molecular docking simulations was highly consistent with the results observed via truncation, single base mutation, and circular dichroism experiments. The fluorescence assay revealed limits of detection and quantification of 4.1 and 12 nM for the aptamer, respectively. Comparing our developed aptasensor with LC-MS/MS methodology revealed that it could detect verrucarin A levels in phosphate-buffered saline and dust extracts with robust precision and consistency. Our findings provide insight for future studies exploring interaction mechanisms with intended targets and practical sensing applications, such as point-of-care detection of verrucarin A. © 2024 American Chemical Society.