Comparison of different strategies to select aptamers against a transmembrane protein target.
Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.
Pestourie
Carine
C
CEA, DSV, DRM, Service Hospitalier Frédéric Joliot, INSERM U803, Orsay, France.
Cerchia
Laura
L
Gombert
Karine
K
Aissouni
Youssef
Y
Boulay
Jocelyne
J
De Franciscis
Vittorio
V
Libri
Domenico
D
Tavitian
Bertrand
B
Ducongé
Frédéric
F
D019943:Amino Acid Substitution; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D003432:Cross-Linking Reagents; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016716:PC12 Cells; D012150:Polymorphism, Restriction Fragment Length; D011485:Protein Binding; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D052156:SELEX Aptamer Technique / Q000379:methods*
D052157:Aptamers, Nucleotide; D003432:Cross-Linking Reagents; D008024:Ligands; D008565:Membrane Proteins; D011994:Recombinant Proteins; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human