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DNA-encoded library (DEL) is an efficient high-throughput screening technology platform in drug discovery and is also gaining momentum in academic research. Today, the majority of DELs are assembled and encoded with double-stranded DNA tags (dsDELs) and has been selected against numerous biological targets; however, dsDELs are not amendable to some of the recently developed selection methods, such as the cross-linking-based selection against immobilized targets and live-cell-based selections, which require DELs encoded with single-stranded DNAs (ssDELs). Herein, we present a simple method to convert dsDELs to ssDELs using exonuclease digestion without library redesign and resynthesis. We show that dsDELs could be efficiently converted to ssDELs and used for affinity-based selections either with purified proteins or on live cells.

Converting Double-Stranded DNA-Encoded Libraries (DELs) to Single-Stranded Libraries for More Versatile Selections