Solid-phase DNA-encoded library (DEL) synthesis is a next-generation drug discovery technology with powerful activity-based and cellular lead identification capabilities. Solid-phase DELs combine the one-bead-one-compound approach with DNA encoding to furnish beads that display multiple copies of photocleavable library members and DNA encoding tags. Sequential chemical synthesis and enzymatic DNA ligation reactions yield an encoded library in which individual library members are physically isolable, enabling various high-throughput screening modalities. This advancement from on-DNA synthesis, in which small molecules are directly attached to their DNA-encoding tags, decouples the library member from the steric bulk of the DNA tag, which prevents biased binding to a target. Here we provide step-by-step instructions for solid-phase DEL synthesis, incorporating all of our most recent quality control innovations to ensure robust library production. The protocol begins with on-bead synthesis of a linker containing a spectroscopic handle for chromatographic analysis, an ionization enhancer for mass spectrometry and an alkyne for installation of DNA encoding sites via copper-catalyzed azide-alkyne cycloaddition click chemistry. Coupling of a photocleavable linker before library synthesis enables compound liberation from the bead for activity-based screening. Powerful combinatorial split-and-pool parallel synthesis tactics transform modest collections of small-molecule building blocks into large DELs of all possible building block combinations. Post synthesis, decoding and mass analysis of single DEL beads as well as whole-library deep sequencing provides rigorous chemical and bioinformatic quality control and establishes suitability for screening. The solid-phase chemistry is highly accessible: expertise in chemical synthesis is not necessary and solid-phase synthesis apparatus is routinely available in molecular biology laboratories. This procedure requires ~1 month to complete.
© 2025. Springer Nature Limited.
