UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, 06120 Halle, Germany.
UFZ-Helmholtz Centre for Environmental Research, Department of Environmental and Biotechnology Centre, 04318 Leipzig, Germany.
Int J Mol Sci. 2018 Feb 24;19(2):642. doi: 10.3390/ijms19020642.
New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of . In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of = 20 ± 1 nM.
通过将下一代测序 (NGS) 应用于先前选择的适体库，鉴定出了新的、尚未发现的适体 A 蛋白。该库是在使用 FluMag-SELEX 程序进行的经典 SELEX（配体系统进化的指数富集）实验中获得的，随后进行了克隆和 Sanger 测序。从 Sanger 序列库中鉴定出唯一的蛋白 A 结合适体 PA#2/8，并且能够结合完整的 细胞。在这项研究中，我们通过使用高通量 NGS 方法和数据分析对相同的适体库进行重新测序，扩展了 SELEX 结果。比较了这两个数据池。它们证实了选择了高度复杂和异质的寡核苷酸库，并且一致显示出高孤儿含量以及某些序列组的相对频率相似。但与 Sanger 数据池相比，NGS 池明显由一个包含已知蛋白 A 结合适体 PA#2/8 的序列组主导，该序列组是该组中最常见的序列。此外，我们在 NGS 池中发现了两个新的序列组，分别由 PA-C10 和 PA-C8 表示，它们对蛋白 A 也具有高度特异性。比较亲和性研究揭示了这些适体之间的差异，并证实 PA#2/8 仍然是所选适体库中最有效的序列，其亲和力达到低纳摩尔范围，= 20 ± 1 nM。
