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Development of DNA aptamers using Cell-SELEX

Abstract

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 °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 3 months.

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Figure 1: Schematic representation of DNA aptamer selection using the cell-SELEX strategy.
Figure 2: Agarose gel electrophoresis image showing the products of the various cycles of selected DNA library amplification.
Figure 3: Agarose electrophoresis gel image of preparative selected DNA library pool, confirming the efficiency of amplification.
Figure 4: Agarose gel electrophoresis image showing products of cycle optimization of the second selected DNA library pool amplification.
Figure 5: Flow cytometry binding assay to assess the binding of initially selected DNA pools.
Figure 6: Flow cytometry binding assay.
Figure 7: Agarose gel electrophoresis image to assess the PCR-amplified selected DNA pool for cloning.
Figure 8
Figure 9: Binding assay of FITC-labeled potential aptamer candidates using flow cytometry.
Figure 10: Typical apparent dissociation curve of aptamer-cell interaction as analyzed by Sigma Plot.

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Acknowledgements

The authors would like to acknowledge many of our coworkers whose work is reported here. This work has been supported over the years by US National Institutes of Health and National Science Foundation and Florida State Biomedical Program grants. It is also supported by China 2009ZX10004-312 and a 985 project grant.

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D.S. and W.T. originated the concept of cell-SELEX; K.S., D.S., M.O., X.X. and W.T. were responsible for the experimental results and procedures of the project. K.S. wrote most of the manuscript, but all authors contributed to the writing and revision of the manuscript.

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Correspondence to Weihong Tan.

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The authors declare no competing financial interests.

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Sefah, K., Shangguan, D., Xiong, X. et al. Development of DNA aptamers using Cell-SELEX. Nat Protoc 5, 1169–1185 (2010). https://doi.org/10.1038/nprot.2010.66

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