Scoville Delia J, Uhm Tae Kyu Brian, Shallcross Jamie A, Whelan Rebecca J
Department of Chemistry and Biochemistry, Oberlin College, 119 Woodland Street, Oberlin, OH 44074, USA.
J Nucleic Acids. 2017;2017:9879135. doi: 10.1155/2017/9879135. Epub 2017 Feb 9.
CA125 is a mucin glycoprotein whose concentration in serum correlates with a woman's risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target.
CA125是一种粘蛋白糖蛋白，其血清浓度与女性患卵巢癌的风险相关，并且在已确诊患者中还可指示对治疗的反应。在患者样本中准确检测这种大型复杂蛋白具有重大临床意义。我们认为，开发对CA125具有亲和力的核酸适体可能会带来强大的新诊断工具。在此，我们报告了使用单步指数富集配体系统进化技术（One-Pot SELEX）分离对CA125具有亲和力的单链DNA适体，随后对所选寡核苷酸进行高通量测序。这种数据丰富的方法与生物信息学工具相结合，能够对整个筛选过程进行表征。使用荧光各向异性和亲和探针毛细管电泳评估了四种适体候选物的结合亲和力。发现两种适体CA125_1和CA125_12（均无引物）可结合临床相关浓度下的蛋白质靶标。镁离子的存在对结合有不同影响，CA125_1的结合需要镁离子，而镁离子会消除CA125_12的结合。总之，单步指数富集配体系统进化技术被发现是一种很有前景的筛选方法，能够产生针对临床重要蛋白质靶标的DNA适体。
