Thiel William H, Giangrande Paloma H
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA; The François M. Abboud Cardiovascular Research Center, University of Iowa, Iowa City, IA 52242, USA.
Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA; The François M. Abboud Cardiovascular Research Center, University of Iowa, Iowa City, IA 52242, USA; The Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA 52242, USA; The Molecular and Cell Biology Program, University of Iowa, Iowa City, IA 52242, USA.
Methods. 2016 Jul 1;103:180-7. doi: 10.1016/j.ymeth.2016.03.005. Epub 2016 Mar 10.
The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization.
指数富集配体系统进化（SELEX）过程能够从复杂核酸文库中富集对靶分子具有特异性的DNA或RNA适配体。SELEX过程已从使用重组靶蛋白在体外鉴定适配体转变为基于细胞的方法（细胞SELEX），其中靶标在细胞表面表达。细胞SELEX的一个主要优点是靶分子保持天然构象。此外，细胞SELEX可通过对患病细胞与健康细胞进行筛选来发现新型治疗生物标志物。然而，细胞SELEX的一个问题是，对筛选中鉴定出的单个适配体进行测试既费力、耗时又昂贵。在细胞上筛选适配体结合和内化最常用的方法是流式细胞术和定量PCR（qPCR）。虽然流式细胞术可以直接评估荧光标记的适配体与靶标的结合，但它需要大量起始材料且不易扩展。基于qPCR的方法高度灵敏，但由于样品处理步骤的数量，实验间的变异性不可忽略。在此，我们描述了一种基于细胞的适配体荧光结合和内化（AFBI）测定法。该测定法所需试剂最少，实验步骤/操作较少，从而能够同时快速筛选许多适配体和条件，并直接定量适配体的结合和内化。
