title: Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection.
abstract: The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY 14853,USA.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Ozer
authors.forename: Brian S
authors.initials: BS
authors.lastname: White
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
authors.forename: David
authors.initials: D
authors.lastname: Shalloway
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D008024:Ligands; D015233:Models, Statistical; D010455:Peptides / Q000378:metabolism; D052156:SELEX Aptamer Technique
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D010455:Peptides