Development of DNA aptamers using Cell-SELEX
Development of DNA aptamers using Cell-SELEX
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DOI：
10.1038/nprot.2010.66
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Tan, Weihong
中科院分区：
文献类型：
--
作者：
Sefah, Kwame;Shangguan, Dihua;Tan, Weihong
在过去的二十年里，高亲和力的核酸适配子已经被开发出来，用于各种纯分子和复杂系统，如活细胞。从概念上讲，适配子是通过进化过程开发的，随着选择的进行，具有能够与感兴趣的靶结合的特定构象的序列出现并主导池。这项名为CELL-SELEX(通过指数富集法进行配体的系统进化)的协议是一种可以产生能够与感兴趣的细胞类型特异结合的DNA适配子的方法。通常，癌细胞系被用作产生适配子的靶点，这种适体可以将该细胞类型与其他癌症或正常细胞区分开来。单链DNA(SsDNA)文库池与靶细胞孵育。通过在95℃加热细胞-DNA复合体，然后离心，洗掉非结合序列，并从细胞中恢复结合序列。回收的池与对照细胞系孵育，以筛选出与靶标和对照上的共同分子结合的序列，导致靶标的特定结合物浓缩。用异硫氰酸荧光素标记的正义和生物素标记的反义引物，通过聚合酶链式反应扩增结合序列。接下来是去除反义链，为后续几轮选择生成一个单链DNA池。通过流式细胞仪结合分析来监测所选择的池的浓缩，与未选择的DNA文库相比，所选择的池具有更强的荧光。从设计寡核苷酸到对选定的池进行浓缩，整个过程需要大约3个月的时间。
In the past two decades, high-affinity nucleic acid aptamers have been developed for a wide variety of pure molecules and complex systems such as live cells. conceptually, aptamers are developed by an evolutionary process, whereby, as selection progresses, sequences with a certain conformation capable of binding to the target of interest emerge and dominate the pool. this protocol, cell-SELEX (systematic evolution of ligands by exponential enrichment), is a method that can generate DNA aptamers that can bind specifically to a cell type of interest. commonly, a cancer cell line is used as the target to generate aptamers that can differentiate that cell type from other cancers or normal cells. a single-stranded DNA (ssDNA) library pool is incubated with the target cells. nonbinding sequences are washed off and bound sequences are recovered from the cells by heating cell-DNA complexes at 95 degrees C, followed by centrifugation. the recovered pool is incubated with the control cell line to filter out the sequences that bind to common molecules on both the target and the control, leading to the enrichment of specific binders to the target. Binding sequences are amplified by PCR using fluorescein isothiocyanate-labeled sense and biotin-labeled antisense primers. this is followed by removal of antisense strands to generate an ssDNA pool for subsequent rounds of selection. the enrichment of the selected pools is monitored by flow cytometry binding assays, with selected pools having increased fluorescence compared with the unselected DNA library. the procedure, from design of oligonucleotides to enrichment of the selected pools, takes similar to 3 months.