On-Bead Screening of Combinatorial Libraries: Reduction of Nonspecific Binding by Decreasing Surface Ligand Density

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) wa...