Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, Portsmouth, UK.
Methods Mol Biol. 2023;2633:145-161. doi: 10.1007/978-1-0716-3004-4_12.
Nucleic acid aptamers are short sequences of single-stranded (ss) DNA or RNA that fold into a three-dimensional shape with useful binding properties. Traditionally, these properties have included specific recognition and binding of ions, small-molecules, proteins, and enzyme targets. Increasingly though, aptamers are being raised against complex subcellular or cellular targets. These broader-affinity aptamers can be usefully employed for detection, labeling, or therapeutic targeting of intact/living cells, whether prokaryotic or eukaryotic. Aptamers are usually developed from a random-sequence oligonucleotide library by repeated rounds of selection and amplification, a process named "systematic evolution of ligands by exponential enrichment" (SELEX). We describe here a widely applicable cell-SELEX method for raising aptamers against bacteria, using Escherichia coli strain HB101 as an example. Our cell-SELEX method uses a cycle of four stages: (1) incubation of a fluorescently labeled random-sequence ssDNA library with bacterial cells; (2) separation of cell-associated ssDNA from free ssDNA; (3) amplification of bound ssDNA by PCR, and (4) use of lambda-exonuclease to selectively regenerate ssDNA for further rounds of selection.
核酸适体是短序列的单链 (ss) DNA 或 RNA，可折叠成具有有用结合特性的三维形状。传统上，这些特性包括对离子、小分子、蛋白质和酶靶标的特异性识别和结合。然而，越来越多的适体针对复杂的亚细胞或细胞靶标被提出。这些更广泛亲和力的适体可用于检测、标记或治疗原代/活细胞，无论是原核细胞还是真核细胞。适体通常通过重复的选择和扩增循环从随机序列寡核苷酸文库中开发出来，这个过程被称为“指数富集的配体系统进化”（SELEX）。我们在这里描述了一种广泛适用的用于针对细菌筛选适体的细胞 SELEX 方法，以大肠杆菌 HB101 菌株为例。我们的细胞 SELEX 方法使用四个阶段的循环：（1）用荧光标记的随机序列 ssDNA 文库与细菌细胞孵育；（2）将细胞结合的 ssDNA 与游离的 ssDNA 分离；（3）通过 PCR 扩增结合的 ssDNA；（4）使用 λ-核酸外切酶选择性地再生 ssDNA 以进行进一步的选择循环。
