Crosslinking of DNA-linked ligands to target proteins for enrichment from DNA-encoded libraries

Abstract

Achieving sufficient enrichment of ligands from DNA-encoded libraries for detection can be difficult, particularly for low affinity ligands within highly complex libraries. To address this challenge, we present an approach for crosslinking DNA-linked ligands to target proteins using electrophilic or photoreactive groups. The approach involves the teathering of a ssDNA oligonucleotide to a DNA-encoded molecule to enable attachment of a reactive group post-synthetically via DNA hybridization. Crosslinking traps ligand-protein complexes while in solution and allows for stringent washing conditions to be applied in the subsequent purification. Five reactive groups (tosyl, NHS ester, sulfonyl fluoride, phenyl azide, and diazirine) were tested for crosslinking efficiency and specificity with three DNA-linked ligands to their target proteins. In a model selection, crosslinking resulted in improved enrichment of both high and a low affinity ligands in comparison to a selection with a solid-phase immobilized protein.

DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information

“…The use of photo-crosslinking methods [Figure 7d] for the screening of DNA-encoded libraries has also been proposed, with encouraging experimental results (69–70). These approaches can be performed in solution and may avoid certain artifacts, that could potentially arise from the immobilization of target proteins on solid supports.…”

Exploring the Lower Limit of Individual DNA-Encoded Library Molecules in Selection

“…The binding process is determined by the reversible interaction of the small molecules with targets. In some studies, the DEL molecules were with covalent warheads, 22 cross-linking ligands, 23 or target proteins conjugated with complementary DNA tags 24 ; the selection strategies in these cases were very different, and our conclusion does not apply.…”

Application of a Substrate-Mediated Selection with c-Src Tyrosine Kinase to a DNA-Encoded Chemical Library

“…High specific activity, which is a critical requirement for affinity-mediated selections, can be problematic for protein kinases. Our attempts at affinity-based selection with an immobilized c-Src were unsuccessful for either a DNA-linked LYNtide or SrcDEL10 , which we presume was due to low specific activity of the enzyme as selections with ligands of low micromolar affinity are generally successful [63].…”