Mayer Günter, Höver Thomas
University of Bonn, Bonn, Germany.
Methods Mol Biol. 2009;535:19-32. doi: 10.1007/978-1-59745-557-2_2.
Aptamers are single-stranded nucleic acids that bind specifically to a target molecule and thus often inhibit target-associated biological functions. Aptamers have been described for a series of target molecules including peptides, proteins, and even living cells. Besides RNA and 20-modified RNA molecules also ssDNA molecules can be subjected to in vitro selection protocols aiming at the enrichment of ssDNA aptamers. ssDNA aptamers can be selected using the SELEX procedure (systematic enrichment of ligands by exponential amplification) from libraries of randomized single-stranded DNA with a diversity of up to 10(16) different molecules. In repetitive selection cycles, the library is incubated with the target of choice and separation of non-binding sequences from bound sequences is achieved by distinct separation methods. The bound molecules are specifically eluted and amplified, thus representing the starting library for the next cycle. Thereby, an enriched population of aptamers is evolved. Here we describe a generalized in vitro selection experiment aiming at the enrichment of ssDNA aptamers using biotinylated target molecules. This procedure allows the application of streptavidin-biotin chemistry to separate bound from unbound DNA species during the selection process.
适配体是单链核酸，能特异性结合靶分子，因此常抑制与靶标相关的生物学功能。已针对一系列靶分子（包括肽、蛋白质甚至活细胞）描述了适配体。除了RNA和20种修饰的RNA分子外，单链DNA分子也可采用体外筛选方案，以富集单链DNA适配体。可使用SELEX程序（通过指数扩增系统富集配体）从多样性高达10（16）种不同分子的随机单链DNA文库中筛选单链DNA适配体。在重复的筛选循环中，将文库与选定的靶标一起孵育，并通过不同的分离方法将未结合序列与结合序列分离。结合的分子被特异性洗脱并扩增，从而作为下一轮循环的起始文库。由此，进化出一个富集的适配体群体。在此，我们描述了一个通用的体外筛选实验，旨在使用生物素化靶分子富集单链DNA适配体。该程序允许在筛选过程中应用链霉亲和素-生物素化学方法分离结合和未结合的DNA种类。
