Max F Perutz Laboratories, Department of Biochemistry, University of Vienna, Vienna, Austria.
PLoS One. 2010 Feb 11;5(2):e9169. doi: 10.1371/journal.pone.0009169.
SELEX is a well established in vitro selection tool to analyze the structure of ligand-binding nucleic acid sequences called aptamers. Genomic SELEX transforms SELEX into a tool to discover novel, genomically encoded RNA or DNA sequences binding a ligand of interest, called genomic aptamers. Concerns have been raised regarding requirements imposed on RNA sequences undergoing SELEX selection.
METHODOLOGY/PRINCIPAL FINDINGS: To evaluate SELEX and assess the extent of these effects, we designed and performed a Neutral SELEX experiment omitting the selection step, such that the sequences are under the sole selective pressure of SELEX's amplification steps. Using high-throughput sequencing, we obtained thousands of full-length sequences from the initial genomic library and the pools after each of the 10 rounds of Neutral SELEX. We compared these to sequences obtained from a Genomic SELEX experiment deriving from the same initial library, but screening for RNAs binding with high affinity to the E. coli regulator protein Hfq. With each round of Neutral SELEX, sequences became less stable and changed in nucleotide content, but no sequences were enriched. In contrast, we detected substantial enrichment in the Hfq-selected set with enriched sequences having structural stability similar to the neutral sequences but with significantly different nucleotide selection.
CONCLUSIONS/SIGNIFICANCE: Our data indicate that positive selection in SELEX acts independently of the neutral selective requirements imposed on the sequences. We conclude that Genomic SELEX, when combined with high-throughput sequencing of positively and neutrally selected pools, as well as the gnomic library, is a powerful method to identify genomic aptamers.
SELEX 是一种成熟的体外选择工具，用于分析配体结合核酸序列的结构，这些序列被称为适体。基因组 SELEX 将 SELEX 转化为一种发现与感兴趣配体结合的新型基因组编码 RNA 或 DNA 序列的工具，称为基因组适体。人们对 SELEX 选择过程中对 RNA 序列施加的要求提出了担忧。
方法/主要发现：为了评估 SELEX 并评估这些影响的程度，我们设计并进行了一项中性 SELEX 实验，省略了选择步骤，使得序列仅受到 SELEX 扩增步骤的选择性压力。使用高通量测序，我们从初始基因组文库和 10 轮中性 SELEX 后的每个池获得了数千个全长序列。我们将这些序列与从相同初始文库获得的基因组 SELEX 实验获得的序列进行了比较，但筛选的是与大肠杆菌调节蛋白 Hfq 具有高亲和力结合的 RNA。随着每一轮中性 SELEX，序列的稳定性降低，核苷酸含量发生变化，但没有序列富集。相比之下，我们在 Hfq 选择的集合中检测到了大量的富集，富集的序列具有与中性序列相似的结构稳定性，但核苷酸选择明显不同。
结论/意义：我们的数据表明，SELEX 中的正选择独立于对序列施加的中性选择要求。我们得出结论，基因组 SELEX 与高通量测序的正选择和中性选择池以及基因组文库相结合，是识别基因组适体的强大方法。
