Spiga Fabio M, Maietta Paolo, Guiducci Carlotta
†Institute of Bioengineering, Ecole Polytechnique Féderale De Lausanne (EPFL), Lausanne, Vaud, 1015, Switzerland.
‡Structural Computational Biology Group, Spanish National Cancer Research Centre (CNIO), Madrid, 28029, Spain.
ACS Comb Sci. 2015 May 11;17(5):326-33. doi: 10.1021/acscombsci.5b00023. Epub 2015 Apr 28.
To address limitations in the production of DNA aptamers against small molecules, we introduce a DNA-based capture-SELEX (systematic evolution of ligands by exponential enrichment) protocol with long and continuous randomized library for more flexibility, coupled with in-stream direct-specificity monitoring via SPR and high throughput sequencing (HTS). Applying this capture-SELEX on tobramycin shows that target-specificity arises at cycle number 8, which is confirmed by sequence convergence in HTS analysis. Interestingly, HTS also shows that the most enriched sequences are already visible after only two capture-SELEX cycles. The best aptamers displayed K(D) of approximately 200 nM, similar to RNA and DNA-based aptamers previously selected for tobramycin. The lowest concentration of tobramycin detected on label-free SPR experiments with the selected aptamers is 20-fold smaller than the clinical range limit, demonstrating suitability for small-drug biosensing.
为了解决针对小分子的DNA适配体生产中的局限性，我们引入了一种基于DNA的捕获SELEX（指数富集配体系统进化）方案，该方案具有长且连续的随机文库以提供更大的灵活性，并通过表面等离子体共振（SPR）和高通量测序（HTS）进行在线直接特异性监测。将这种捕获SELEX应用于妥布霉素表明，在第8轮循环时出现了靶标特异性，这在HTS分析中的序列趋同中得到了证实。有趣的是，HTS还表明，仅经过两轮捕获SELEX循环后，最富集的序列就已经可见。最佳适配体的解离常数（K(D)）约为200 nM，与先前为妥布霉素选择的基于RNA和DNA的适配体相似。使用所选适配体进行的无标记SPR实验中检测到的最低妥布霉素浓度比临床范围限值小20倍，证明其适用于小分子药物生物传感。
