In vitro selection using a dual RNA library that allows primerless selection.
In vitro selection using a dual RNA library that allows primerless selection.
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DOI：
10.1093/nar/gkl463
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发表时间：
2006-07-19
影响因子：
14.9
通讯作者：
Klussmann S
中科院分区：
文献类型：
--
作者：
Jarosch F;Buchner K;Klussmann S
通过称为指数富集配体系统进化 (SELEX) 的体外选择过程，从随机寡核苷酸文库中鉴定出高亲和力靶标结合适体。由于 SELEX 过程包括 PCR 扩增步骤，寡核苷酸文库的随机区域需要两侧有两个固定的引物结合序列。这些引物结合位点通常难以截短，因为它们可能是维持适体结构所必需的，甚至可能是靶结合基序的一部分。我们设计了一种新型RNA文库，其携带固定序列，将寡核苷酸限制为部分双链结构，从而最大限度地降低引物结合序列成为靶标结合基序一部分的风险。此外，文库的具体设计（包括使用串联 RNA 聚合酶启动子）允许选择没有任何引物结合序列的寡核苷酸。该文库用于选择生长素释放肽镜像肽的适体。生长素释放肽是生长激素释放和食物摄入的有效刺激剂。选择后，直接以镜像配置合成所识别的适体序列。最终的 44 nt-Spiegelmer 名为 NOX-B11-3，在细胞培养测定中阻断 ghrelin 作用，在 37°C 下的 IC50 为 4.5 nM。
High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C.