Enzymatic Cleavage of Double‐Stranded DNA‐Encoded Libraries (DELs) to Single‐Stranded DELs with Compounds at the 3' End: Its Application in Photo‐crosslinking Selection

DNA‐encoded library (DEL) technology is a crucial tool in pharmaceutical research, rapidly identifying compounds that bind to a target of interest from an extensive pool of compounds. In this study, we propose a new method for generating single‐stranded DELs (ssDELs) with compounds at the 3’ end. The introduction of uniquely designed hairpin‐shaped headpieces containing deoxyuridine (NC‐HP) and the use of a cleavage enzyme facilitate the conversion from double‐stranded DELs (dsDELs) to such ssDELs. Moreover, Klenow fill‐in provides the dsDELs with photo‐crosslinkers covalently linked to the coding region, which exhibit durability even under stringent washing conditions and enable photo‐crosslinking with a high signal‐to‐noise ratio, as also confirmed in cell‐based photo‐crosslinking selections.