Department of Civil and Environmental Engineering and Institute of Construction and Environmental Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
Division of Biomedical Technology, Daegu Gyeongbuk Institute of Science and Technology (DGIST), 333 Techno Jungang-daero, Hyeonpung-eup, Dalseong-gun, Daegu 42988, Republic of Korea.
Anal Chem. 2024 Oct 29;96(43):17262-17270. doi: 10.1021/acs.analchem.4c03307. Epub 2024 Oct 15.
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 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.
适体是一种单链寡核苷酸，作为一种具有高特异性和结合亲和力的化学抗体，可以识别广泛的分子。有效修饰和截断适体可以增强它们与特定靶标的结合亲和力，同时拓宽其在生物传感器等方面的应用。然而，传统的反复试验方法阻碍了这一过程。在这里，我们展示了一种阐明针对室内建筑中霉菌产生的真菌毒素疣孢菌素 A 的单链 DNA 适体结合位点的方法。这种新的 ssDNA 适体表现出 29.5 nM 的结合亲和力，与先前报道的针对小分子的典型适体相比，具有相对较强的亲和力。此外，所选适体对结构相关真菌毒素（即疣孢酚和玉米赤霉烯酮）中的疣孢菌素 A 具有高度特异性。通过分子动力学和分子对接模拟预测的适体结合位点与通过截断、单碱基突变和圆二色性实验观察到的结果高度一致。荧光测定法显示该适体的检测限和定量限分别为 4.1 和 12 nM。将我们开发的适体传感器与 LC-MS/MS 方法进行比较，结果表明它可以在磷酸盐缓冲盐水和粉尘提取物中检测到具有稳健精度和一致性的疣孢菌素 A 水平。我们的研究结果为未来研究与预期靶标相互作用机制和实际传感应用（如即时检测疣孢菌素 A）提供了思路。
