Microfluidic Agarose Microdroplets for DNA-Encoded Chemical Library Screening

Abstract

DNA-encoded library (DEL) technology enables high-throughput small-molecule discovery but is typically performed using purified proteins under in vitro conditions that do not reflect native intracellular environments. Here, we present a microfluidic agarose -droplet platform for cellular-context DEL screening. The porous hydrogel droplets provide mechanically stable yet permeable microenvironments that protect weak protein-ligand interactions while enabling efficient buffer exchange and ligand diffusion. Importantly, mild cell permeabilization within droplets selectively retained chromatin-associated proteins, allowing screening directly in a cellular context. Using BRD4 as a model target, we validated intracellular ligand engagement by fluorescence imaging and super-resolution microscopy. Small-scale DEL screening selectively enriched JQ1 in both bead-based and cell-based formats, and large-scale DEL screening across millions of encoded compounds successfully identified hit molecules by sequencing. This agarose -droplet-based strategy expands DEL technology toward biologically relevant and chromatin-associated targets under near-native conditions.