Selection of DNA‐Encoded Small Molecule Libraries Against Unmodified and Non‐Immobilized Protein Targets

Abstract

The selection of DNA-encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand-induced photo-crosslinking, we show that iterated selections of DNA-encoded libraries can be realized with unmodified and non-immobilized protein targets.

Design, preparation, and selection of DNA-encoded dynamic libraries

“…Second, our method eliminated the requirement of target immobilization and physical washing; therefore, target-induced perturbation of the library equilibrium is better preserved, and unmodified, non-immobilized proteins can be used as targets. 69,70,90 …”

“…Moreover, previous studies require modified and immobilized targets in library selection, which is not compatible with proteins that are difficult to purify or modify, such as membrane proteins. 69,70 Aiming to address these issues, here we report the detailed study of a DEDL system, including library preparation, encoding, selection, hit deconvolution, and notably, a novel “locking” strategy to freeze the equilibrium shift for hit isolation and identification.…”

Novel selection methods for DNA-encoded chemical libraries

“…UV irradiation leads to covalent attachment of the PC-DNA to the target protein, which can be identified by mass spectrometry. The DPAL technique was adapted to identify binders from libraries of small molecules by taking advantage of hybridization between the PC-DNA and the binding ligands' DNA tags [22]. The DNA tags of non-binding small molecules are digested by Exonuclease I, but the hybridized DNA tags of bound molecules are protected from digestion.…”

“…These potential selection methods would not require solid-phase immobilization, washing, or elution steps, but would render the DNA-encoded libraries single-use. Indeed, DPAL, IDUP, and the proximity extension assay all take advantage of protein-small molecule complexes' ability to protect DNA from exonucleases to decrease the signal arising from inactive library members [20,22,28]. Complexes that prevent DNA from interacting with T4 DNA ligase [47] or T7 RNA polymerase [48] could also be adapted to evaluate DNA-encoded chemical libraries.…”

Discovery of a Covalent Kinase Inhibitor from a DNA-Encoded Small-Molecule Library × Protein Library Selection

“…2,3 The effectiveness of these methods is limited by artifactual enrichment of library members that bind the solid support or nonphysiologically relevant forms of target proteins, incomplete knowledge of the biological context necessary for the target to adopt its relevant form, and the inability to simultaneously explore interactions with multiple proteins of interest. Few methods of screening DNA-encoded libraries, such as selections on cell-surface displayed proteins, 4 parallel selections under varied conditions, 3 or the use of photo-crosslinking probes to perform selections on unmodified proteins, 5 have begun to address these limitations.…”