Ulrich Henning, Martins Antonio Henrique B, Pesquero João Bosco
Universidade de São Paulo, São Paulo, Brazil.
Curr Protoc Cytom. 2005 Aug;Chapter 7:Unit 7.28. doi: 10.1002/0471142956.cy0728s33.
Using systematic evolution of ligands by exponential enrichment (SELEX), RNA or DNA molecules are selected from a combinatorial oligonucleotide library by their ability to bind their targets, i.e., cell surface antigens, with affinity and specificity similar to that of monoclonal antibodies. The generation of these high-affinity binders, also denominated aptamers, is carried out in vitro and does not involve animals. Therefore, aptamers can be developed against almost every molecule of biological importance, including toxins and nonimmunogenic targets, against which antibodies cannot be raised. The incorporation of modified pyrimidines resulting in nuclease-resistant RNA aptamers makes them promising candidates for studying protein interactions in vitro and in vivo. DNA aptamers do not need modifications for most applications. The protocols in this unit can be used for the development of fluorescent-tagged RNA or DNA aptamers for any cell surface protein in cytomics analysis.
利用指数富集的配体系统进化技术（SELEX），可从组合寡核苷酸文库中筛选出RNA或DNA分子，这些分子能够以与单克隆抗体相似的亲和力和特异性结合其靶标，即细胞表面抗原。这些高亲和力结合物，也称为适配体，是在体外产生的，不涉及动物。因此，适配体几乎可以针对每一种具有生物学重要性的分子进行开发，包括毒素和无法产生抗体的非免疫原性靶标。引入修饰的嘧啶可产生抗核酸酶的RNA适配体，这使其成为在体外和体内研究蛋白质相互作用的有前景的候选物。对于大多数应用而言，DNA适配体无需修饰。本单元中的方案可用于开发用于细胞组学分析中任何细胞表面蛋白的荧光标记RNA或DNA适配体。
