Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing, 100081, China.
School of Computer Science and Engineering, Central South University, China.
Talanta. 2019 Dec 1;205:120088. doi: 10.1016/j.talanta.2019.06.088. Epub 2019 Jun 27.
Capillary electrophoresis-based systematic evolution of ligands by exponential enrichment (CE-SELEX) is a high-efficient technique for aptamers selection, and has been evolved into many modes. In this study, we obtained the aptamer against bovine lactoferrin (BLF) with high affinity (dissociation constant, K = 20.74 ± 6.89 nM) and good specificity (>1000 folds) using single step CE-SELEX (ssCE-SELEX) mode. In the selection process, ssCE demonstrated high-efficiency selection with bulk K reaching at 0.19 ± 0.04 μM by only two rounds, as compared to capillary zone electrophoresis (CZE) mode with K of 0.39 ± 0.03 μM. Next-generation sequencing (NGS) was performed by two methods of high output (Hiseq) and medium output (Miseq) with different sequencing depths, and their same results of high-frequency sequences confirmed the reliability of the obtained sequences. Through affinity analysis, the primer region and single base mutation (SBM) were observed to affect the sequence structure and to result in affinity change. Besides, molecular dynamics (MD) simulation was performed to validate the binding affinity of the candidates with BLF by analyzing binding sites, interaction forces, and binding free energy. Moreover, BLF detection in milk powder matrices was completed successfully with the optimized CE-aptasensor. The signal response was in a good linear relationship (R = 0.9930) with 4-128 nM of BLF and the detection limit was 1 nM. The obtained results of BLF in four milk powder samples were in an acceptable agreement with the labeled concentrations. This study presented a completed CE based process including aptamers selection, affinity characterization, and detection application, which also validated the high-efficiency selection of ssCE-SELEX mode.
基于毛细管电泳的指数富集配体系统进化（CE-SELEX）是一种高效的适体筛选技术，已经发展出多种模式。本研究采用单步 CE-SELEX（ssCE-SELEX）模式，获得了与牛乳铁蛋白（BLF）具有高亲和力（解离常数，K=20.74±6.89 nM）和良好特异性（>1000 倍）的适体。在筛选过程中，ssCE 仅通过两轮就达到了高效筛选， bulk K 值达到 0.19±0.04 μM，而毛细管区带电泳（CZE）模式的 K 值为 0.39±0.03 μM。下一代测序（NGS）通过高输出（Hiseq）和中输出（Miseq）两种方法进行，测序深度不同，其高频序列的相同结果证实了获得序列的可靠性。通过亲和力分析，观察到引物区域和单碱基突变（SBM）影响序列结构并导致亲和力变化。此外，通过分析结合位点、相互作用力和结合自由能，进行分子动力学（MD）模拟，验证候选物与 BLF 的结合亲和力。此外，还成功地完成了优化后的 CE-适体传感器对奶粉基质中 BLF 的检测。信号响应与 4-128 nM 的 BLF 呈良好的线性关系（R=0.9930），检测限为 1 nM。在四个奶粉样品中 BLF 的测定结果与标记浓度具有良好的一致性。本研究提供了一个完整的基于 CE 的过程，包括适体筛选、亲和力表征和检测应用，也验证了 ssCE-SELEX 模式的高效筛选。
