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 an...

Full description

Saved in:
Bibliographic Details
Published inJournal of combinatorial chemistry Vol. 11; no. 4; pp. 604 - 611
Main Authors Chen, Xianwen, Tan, Pauline H, Zhang, Yanyan, Pei, Dehua
Format Journal Article
LanguageEnglish
Published WASHINGTON American Chemical Society 13.07.2009
Amer Chemical Soc
Subjects
Online AccessGet full text

Cover

Loading…
More Information
Summary: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.
Bibliography:NIH RePORTER
ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:1520-4766
1520-4774
DOI:10.1021/cc9000168