Abstract
High-diversity genetically-encoded combinatorial libraries (108−1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. Synthetic libraries can access broader chemical space, but typically examine only ~ 106 compounds by screening. Here we show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members—a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106–108. These results are then applied to the discovery of p53-like binders to MDM2, and to a family of 3–19 nM-affinity, α/β-peptide-based binders to 14-3-3. An X-ray structure of one of these binders in complex with 14-3-3σ is determined, illustrating the role of β-amino acids in facilitating a key binding contact.
Highlights
High-diversity genetically-encoded combinatorial libraries (108−1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection
While a variety of techniques are used to this end, some of the most common strategies include high-throughput screening (HTS) and fragment-based drug discovery (FBDD)[2] for identification of small molecule binders, and protein engineering strategies[3] for discovery of novel biologics
Given the unique niche of chemical space they occupy, possessing molecular weights in between those of small molecules (
Summary
High-diversity genetically-encoded combinatorial libraries (108−1013 members) are a rich source of peptide-based binding molecules, identified by affinity selection. We show that in-solution affinity selection can be interfaced with nano-liquid chromatography-tandem mass spectrometry peptide sequencing to identify binders from fully randomized synthetic libraries of 108 members—a 100-fold gain in diversity over standard practice. To validate this approach, we show that binders to a monoclonal antibody are identified in proportion to library diversity, as diversity is increased from 106–108. Molecular biology-based selection techniques, such as phage display[14,15] and mRNA display[16], are powerful tools for targetbased discovery of novel peptide binders, thanks in part to their ability to examine enormous libraries (108–1013 members)[17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
