Selection of Aptamers Against Whole Living Cells: From Cell-SELEX to Identification of Biomarkers
Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.
Key wordsAptamer Cell-SELEX Oligonucleotides Biomarkers
This protocol of cell-SELEX was first developed in the lab of Domenico Libri before being further optimized in the lab of Bertrand Tavitian and now in the Neurodegenerative Diseases Laboratory, we thank all our collaborators from these labs for help and fruitful discussions, especially Carine Pestourie, Karine Gombert, Benoit Jego, Isabelle Janssens, Jocelyne Boulay, Mohamed Aissouni, Bertrand Tavitian, and Domenico Libri. We are also grateful to Dr. Rui Sousa (University of Texas, San Antonio) for his generous gift of a T7Y639F RNA polymerase-expressing plasmid. Studies relating to selection of aptamers in our laboratories were supported by grants from the “Agence Nationale pour la Recherche” [projects ANR-RNTS TomoFluo3D, ANR-PNANO nanorings and under the frame of EuroNanoMed (project META)]; the FMT-XCT European program [Grant agreement no. 201792] and the European Molecular Imaging Laboratory (EMIL) network [EU contract LSH-2004-503569].
- 14.Ohuchi, S.P., Ohtsu, T. and Nakamura, Y. (2005) A novel method to generate aptamers against recombinant targets displayed on the cell surface. Nucleic Acids Symp Ser (Oxf), 49:351–352Google Scholar