Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
Analyst. 2017 Oct 23;142(21):4030-4038. doi: 10.1039/c7an00909g.
A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology called polymer-enhanced capillary transient isotachophoresis (PectI). The PectI separation yielded a single peak for the human lung cancer cell line (PC-9) complexed with DNA aptamer candidates, which was effectively separated from a free randomized DNA library peak, ensuring no contamination from free DNA in the PC-9-DNA aptamer complex fraction. The DNA aptamer candidates obtained after a single-round selection employing counter selection with HL-60 were proven to bind selectively and form kinetically stable complexes with PC-9 cells. Interestingly, most aptamer candidates showed high binding ability (K = 70-350 nM) with different extents of binding on the cell surface. These facts proved that a single-round selection for mammalian cells by PectI is feasible to obtain various types of aptamer candidates, which have high-affinity even for non-overexpressed but unique targets on the cell surface in addition to overexpressed targets.
首次成功地使用一种基于毛细管电泳（CE）的方法——聚合物增强毛细管瞬态等速电泳（PectI），对哺乳动物细胞进行了一轮 DNA 适体选择。PectI 分离得到了与人肺癌细胞系（PC-9）与 DNA 适体候选物结合的单一峰，该峰与游离随机 DNA 文库峰有效分离，确保了 PC-9-DNA 适体复合物部分没有游离 DNA 的污染。经过一轮选择，然后用 HL-60 进行反选择，获得的 DNA 适体候选物被证明能与 PC-9 细胞选择性结合，并形成动力学稳定的复合物。有趣的是，大多数适体候选物显示出高的结合能力（K=70-350 nM），在细胞表面的结合程度不同。这些事实证明，通过 PectI 对哺乳动物细胞进行一轮选择是可行的，可以获得各种类型的适体候选物，这些适体候选物除了高表达的靶标外，对细胞表面非高表达但独特的靶标也具有高亲和力。
