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.

Hi‐Fi SELEX: A high‐fidelity digital‐PCR based therapeutic aptamer discovery platform

“…As the hydrodynamic diameter of a random 80‐mer DNA aptamer is ca . 7.9 nm, this surface density permits aptamer screening anywhere within the theoretically preferred 100:1 to 1000:1 aptamer‐to‐target range, while eliminating the possibility of bridging of aptamers between proximal immobilized targets, an effect that has been shown to confound aptamer selection (Ozer et al, ).…”

Development of ssDNA Aptamers for Diagnosis and Inhibition of the Highly Pathogenic Avian Influenza Virus Subtype H5N1

“…Typically, many SELEX processes contain steps for target immobilization to solid supports [29]. However, this can introduce nonspecific binding to the aptamer, limiting the enrichment of most specific aptamers during the SELEX process [30]. Many simple isolation methods for bound aptamers in supernatants have been used generally in recent studies [31,32].…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…In addition, the molecules cannot always be considered point particles as selections may be affected by molecule size and orientation. 36,37 Although some theoretical work has been done to fill these information gaps, [38][39][40][41][42] most parameters remain experimentally unknown, and researchers have adopted simple intuitive schemes to attempt to create competitiveness and stringency during selections, such as gradually increasing the ratio of library to target molecules (i.e., the fold-excess of library to target) and/or reducing the quantity or concentration of target molecules. These selections are typically performed in one of three ways.…”

“…Recent studies have shown that in certain technologies, many of the core and intuitive assumptions of the SELEX model and basic binding kinetics are not well supported by the binding results. 36,37,[60][61][62]66,67,75 This is likely due to the added complexity of the more sophisticated technologies and immobilization schemes used. Selection techniques are currently evaluated primarily on the number of cycles needed and/or on the binding affinity of the resulting aptamer.…”