Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota, 55455, United States.
Anal Chem. 2013 Nov 19;85(22):10761-70. doi: 10.1021/ac401875h. Epub 2013 Nov 1.
Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) is a powerful technique for isolating aptamers for various targets, from large proteins to small peptides with molecular weights of several kilodaltons. One of the unique characteristics of CE-SELEX is the relatively high heterogeneity of the ssDNA pools that remains even after multiple rounds of selection. Enriched sequences or highly abundant oligonucleotide motifs are rarely reported in CE-SELEX studies. In this work, we employed 454 pyrosequencing to profile the evolution of an oligonucleotide pool through multiple rounds of CE-SELEX selection against the target recombinant human vascular endothelial growth factor 165 (rhVEGF165). High throughput sequencing allowed up to 3 × 10(4) sequences to be obtained from each selected pool and compared to the unselected library. Remarkably, the highest abundance contiguous sequence (contig) was only present in 0.8% of sequences even after four rounds of selection. Closer analyses of the most abundant contigs, the top 1000 oligonucleotide fragments, and even the eight original FASTA files showed no evidence of prevailing motifs in the selected pools. The sequencing results also provided insight into why many CE-SELEX selections obtain pools with reduced affinities after many rounds of selection (typically >4). Preferential amplification of a particular short polymerase chain reaction (PCR) product allowed this nonbinding sequence to overtake the pool in later rounds of selection suggesting that further refinement of primer design or amplification optimization is necessary. High affinity aptamers with 10(-8) M dissociation constants for rhVEGF165 were identified. The affinities of the higher abundance contigs were compared with aptamers randomly chosen from the final selection pool using affinity capillary electrophoresis (ACE) and fluorescence polarization (FP). No statistical difference in affinity between the higher abundance contigs and the randomly chosen aptamers was observed, supporting the premise that CE-SELEX selects a uniquely heterogeneous pool of high affinity aptamers.
毛细管电泳-指数富集配体系统进化（CE-SELEX）是一种强大的技术，可用于分离针对各种靶标的适体，从大蛋白质到分子量为几千道尔顿的小肽。CE-SELEX 的一个独特特征是 ssDNA 池的相对较高异质性，即使经过多轮选择后仍然存在。在 CE-SELEX 研究中很少报道富集的序列或高度丰富的寡核苷酸基序。在这项工作中，我们使用 454 焦磷酸测序技术来分析针对靶标重组人血管内皮生长因子 165（rhVEGF165）的寡核苷酸池在多轮 CE-SELEX 选择过程中的进化。高通量测序允许从每个选择池获得多达 3×10(4)个序列，并与未选择的文库进行比较。值得注意的是，即使经过四轮选择，最高丰度连续序列（contig）仅存在于 0.8%的序列中。对最丰富的 contig、前 1000 个寡核苷酸片段，甚至是最初的 8 个 FASTA 文件进行更仔细的分析，没有证据表明选择池中有流行的基序。测序结果还深入了解了为什么许多 CE-SELEX 选择在多轮选择后获得亲和力降低的池（通常>4）。特定短聚合酶链反应（PCR）产物的优先扩增允许该非结合序列在以后的选择轮次中超过池，表明需要进一步改进引物设计或扩增优化。确定了对 rhVEGF165 的解离常数为 10(-8) M 的高亲和力适体。使用亲和毛细管电泳（ACE）和荧光偏振（FP）比较了高丰度 contig 的亲和力与从最终选择池中随机选择的适体。没有观察到高丰度 contig 和随机选择的适体之间的亲和力存在统计学差异，这支持了 CE-SELEX 选择独特的高亲和力适体异质池的前提。
