School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing 100081, China; State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
Beijing Centre for Physical and Chemical Analysis, Beijing 100089, China.
J Chromatogr A. 2014 Sep 5;1358:269-76. doi: 10.1016/j.chroma.2014.06.079. Epub 2014 Jul 1.
For whole-cell aptamers selection, cells surface situation has great impact on single-stranded (ssDNA) binding and aptamers selection. In this work, both Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were as cells targets, their interaction with ssDNA library were evaluated based on capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) with UV and LIF detection. Our results demonstrated that protoplasts without cells wall had apparently stronger interaction with ssDNA library than bacteria, the protoplasts-ssDNA complex could be observed clearly with CZE-LIF. Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol, formaldehyde and glutaraldehyde) showed binding difference with ssDNA library, which could be identified with ACE-UV. Binding constants indicated the interaction of E. coli with ssDNA library were in the order of E. coli protoplasts>methanol (ethanol) treated E. coli>formaldehyde (glutaraldehyde) treated E. coli≈E. coli. Above results suggest that cells surface situation determines their binding affinity with ssDNA, which should be considered in whole-cell aptamers selection and aptamers further application. Capillary electrophoresis is a preferable technique for interaction evaluation of composite targets binding with ssDNA library.
用于全细胞适体筛选的方法中,细胞表面情况对单链 DNA(ssDNA)的结合和适体筛选有重大影响。在这项工作中,嗜酸乳杆菌和大肠杆菌及其原生质体都被用作细胞靶标,基于毛细管区带电泳(CZE)和带有 UV 和 LIF 检测的亲和毛细管电泳(ACE)评估了它们与 ssDNA 文库的相互作用。我们的结果表明,没有细胞壁的原生质体与 ssDNA 文库的相互作用明显强于细菌,CZE-LIF 可清晰观察到原生质体-ssDNA 复合物。此外,用四种有机溶剂(甲醇、乙醇、甲醛和戊二醛)预处理的大肠杆菌与 ssDNA 文库表现出结合差异,可通过 ACE-UV 进行鉴定。结合常数表明,大肠杆菌与 ssDNA 文库的相互作用顺序为大肠杆菌原生质体>甲醇(乙醇)处理的大肠杆菌>甲醛(戊二醛)处理的大肠杆菌≈大肠杆菌。以上结果表明,细胞表面情况决定了它们与 ssDNA 的结合亲和力,这在全细胞适体筛选和适体的进一步应用中应予以考虑。毛细管电泳是评估复合靶标与 ssDNA 文库相互作用的一种较好技术。