Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing.
Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing.
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DOI：
10.1007/s00216-015-8665-7
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发表时间：
2015-09
影响因子：
4.3
通讯作者：
Whelan RJ
中科院分区：
文献类型：
--
作者：
Eaton RM;Shallcross JA;Mael LE;Mears KS;Minkoff L;Scoville DJ;Whelan RJ
癌症生物标志物的新型亲和探针的开发可能使检测和治疗癌症的分析方法得到有力的改进。在这份报告中，我们描述了我们使用毛细管电泳（CE）作为分离机制的过程中选择DNA适体与卵巢癌生物标志物HE4的亲和力。我们在Illumina平台上使用高通量测序，而不是常规使用克隆和测序作为适体选择过程的最后一步。这种数据丰富的方法，结合生物信息学管道的基础上免费提供的计算工具，使整个选择过程，而不仅仅是其终点的特点。亲和探针CE和荧光各向异性测定证明了通过这种生物信息学方法鉴定的一组适体候选物的结合亲和力。在Illumina平台上对候选适体群体进行测序，从而能够表征在多个SELEX轮中选择适体的过程。生物信息学工具用于鉴定所选适体的富集，并基于序列和结构相似性分组为簇。可以智能地选择测序适体的子集用于体外测试。
The development of novel affinity probes for cancer biomarkers may enable powerful improvements in analytical methods for detecting and treating cancer. In this report, we describe our use of capillary electrophoresis (CE) as the separation mechanism in the process of selecting DNA aptamers with affinity for the ovarian cancer biomarker HE4. Rather than the conventional use of cloning and sequencing as the last step in the aptamer selection process, we used high-throughput sequencing on an Illumina platform. This data-rich approach, combined with a bioinformatics pipeline based on freely available computational tools, enabled the entirety of the selection process—and not only its endpoint—to be characterized. Affinity probe CE and fluorescence anisotropy assays demonstrate the binding affinity of a set of aptamer candidates identified through this bioinformatics approach. A population of candidate aptamers is sequenced on an Illumina platform, enabling the process by which aptamers are selected over multiple SELEX rounds to be characterized. Bioinformatics tools are used to identify enrichment of selected aptamers and groupings into clusters based on sequence and structural similarity. A subset of sequenced aptamers may be intelligently chosen for in vitro testing.