On-bead screening of combinatorial libraries: reduction of nonspecific binding by decreasing surface ligand density

Abstract

On-bead screening of one-bead-one-compound (OBOC) libraries provides a powerful method for the rapid identification of active compounds against molecular or cellular targets. However, on-bead screening is susceptible to interference from nonspecific binding, which results in biased screening data and false positives. In this work, we have found that a major source of nonspecific binding is derived from the high ligand loading on the library beads, which permits a macromolecular target (e.g., a protein) to simultaneously interact with multiple ligands on the bead surface. To circumvent this problem, we have synthesized a phosphotyrosyl (pY)-containing peptide library on spatially segregated TentaGel microbeads, which feature a 10-fold reduced peptide loading on the bead surface but a normal peptide loading in the bead interior. The library was screened against a panel of 10 Src homology 2 (SH2) domains including those of Csk and Fyn kinases and adaptor protein SLAP, and the specific recognition motif(s) was successfully identified for each of the domains. In contrast, when the SH2 domains were screened against a control library that contained unaltered (high) ligand loading at the bead surface, six of them exhibited varying degrees of sequence biases, ranging from minor perturbation in the relative abundance of different sequences to the exclusive selection of false positive sequences that have no measurable affinity to the target protein. These results indicate that reduction of the ligand loading on the bead surface represents a simple, effective strategy to largely eliminate the interference from nonspecific binding, while preserving sufficient amounts of materials in the bead interior for compound identification. This finding should further expand the utility of OBOC libraries in biomedical research.

Figure 1

Scheme showing the different mechanisms by which a macromolecular target (e.g., protein) binds to a bead containing immobilized low-molecular-weight ligands (e.g., peptides). (a) Nonspecific binding of a protein containing a negatively charged surface patch (either near or remote from the specific ligand-binding site) to a bead containing positively charged, high-density ligands via multidentate charge-charge interactions (avidity effect), giving rise to false positive beads. (b) Biased binding of a protein to a bead containing a high loading of low-affinity ligands and the weak specific interaction is enhanced by charge-charge interactions between the protein and neighboring ligands. (c) Binding of a protein to a reduced-density bead through high-affinity, specific interaction between the protein and a single immobilized ligand molecule.

Scheme 1

Synthesis of Reduced-Density pY Peptide Library^a

Figure 3

A comparison of the peptide sequences selected from the HD (a) vs LD library (b) against the Fyn SH2 domain. The histograms represent the amino acids identified at each position from pY-2 to pY+3. Number of occurrence on the z axis represents the percentage of selected sequences that contain a particular amino acid at a certain position. M, Nle; C, Abu.

Figure 4

SPR sensograms showing the binding properties of the Fyn SH2 domain to peptides selected from HD (RQpYFRR) vs LD libraries (AYpYAEI). The peptides were biotinylated and immobilized onto a streptavidin-coated sensorchip and the Fyn SH2 domain (5 μM) was flowed over the chip surface. RU, response units.

Figure 5

A comparison of the peptide sequences selected from the HD (a) vs LD library (b) against the SLAP SH2 domain. The histograms represent the amino acids identified at each position from pY-2 to pY+3. Occurrence on the z axis represents the percentage of selected sequences that contain a particular amino acid at a certain position. M, Nle; C, Abu.