Department of Chemistry, National University of Singapore, Republic of Singapore, Singapore.
Electrophoresis. 2010 Jun;31(12):2055-62. doi: 10.1002/elps.200900543.
High-affinity aptamers for important signal transduction proteins, i.e. Cdc42-GTP, p21-activated kinase1 (PAK1) and MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) alpha were successfully selected in the low micro- to nanomolar range using non-systematic evolution of ligands by exponential enrichment (SELEX) with at least three orders of magnitude enhancement from their respective bulk affinity of naïve DNA library. In the non-SELEX procedure, CE was used as a highly efficient affinity method to select aptamers for the desired molecular target through a process that involved repetitive steps of partitioning, known as non-equilibrium CE of equilibrium mixtures with no PCR amplification between successive steps. Various non-SELEX conditions including the type, concentration and pH of the run buffer were optimized. Other considerations such as salt composition of selection buffer, protein concentration and sample injection size were also studied for high stringency during selection. After identifying the best enriched aptamer pool, randomly selected clones from the aptamer pool were sequenced to obtain the individual DNA sequences. The dissociation constants (K(d)) of these sequences were in the low micromolar to nanomolar range, indicating high affinity to the respective proteins. The best binders were also subjected to sequence alignment to generate a phylogenetic tree. No significant consensus region based on approximately 50 sequences for each protein was observed, suggesting the high efficiency of non-SELEX for the selection of numerous unique sequences with high selectivity.
高亲和力适体对于重要的信号转导蛋白，如 Cdc42-GTP、p21 激活激酶 1（PAK1）和 MRCK（肌萎缩性侧索硬化症激酶相关 Cdc42 结合激酶）α，通过指数富集的非系统进化配体（SELEX），从其各自的天然 DNA 文库的体相亲和力中至少提高了三个数量级，成功地在低微摩尔至纳摩尔范围内进行了选择。在非 SELEX 过程中，CE 被用作一种高效的亲和力方法，通过一个涉及重复分配步骤的过程，即没有 PCR 扩增的平衡混合物的非平衡 CE，来选择所需分子靶标的适体。优化了各种非 SELEX 条件，包括运行缓冲液的类型、浓度和 pH 值。还研究了选择缓冲液的盐组成、蛋白质浓度和样品注入大小等其他因素，以在选择过程中保持高严格性。在确定最佳富集适体池后，从适体池中随机选择克隆进行测序，以获得各个 DNA 序列。这些序列的解离常数（Kd）在低微摩尔至纳摩尔范围内，表明对各自蛋白质具有高亲和力。最佳结合物也进行了序列比对，以生成系统发育树。对于每种蛋白质的大约 50 个序列，没有观察到基于显著共识区域的序列，这表明非 SELEX 在选择具有高选择性的大量独特序列方面具有高效率。
