Yoojin Kim ,  Hayeon Kim ,  Jinhui Hong ,  Minseo Kang ,  Jaeyoung Bae ,  Sangyoon Ko ,  Minjae Kim ,  Byumseok Koh ,  Hakjoong Kim ,  Sang-Hee Shim ,  Kyubong Jo

bioRxiv - Bioengineering 

DOI: 10.64898/2026.02.15.706034

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 microdroplet 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 microdroplet based strategy expands DEL technology toward biologically relevant and chromatin-associated targets under near-native conditions.

Summary

This work introduces a microfluidic agarose µ-droplet platform for DNA-encoded chemical library (DECL/DEL) screening against intracellular targets, with validation on BRD4—a nuclear epigenetic reader protein. The system encapsulates single cells or target-coated magnetic beads in monodisperse ~100 µm agarose droplets via flow-focusing microfluidics; the low-gelling-temperature (LGT) agarose forms a porous hydrogel upon cooling, permitting rapid diffusion of DNA-encoded small molecules while preserving intracellular architecture and protein–bead complexes. Permeabilization enables controlled probe access, and super-resolution Exchange-PAINT imaging confirms nanoscale colocalization of JQ1-BP with GFP-BRD4 in nuclear nanoclusters.

Highlights

• Agarose µ-droplets enable gravity-assisted washing, shear protection, and uniform molecular diffusion.
• Two-color Exchange-PAINT with orthogonal R2/R6 docking strands validates specific intracellular target engagement.
• DEL screening yields target-specific enrichment: JQ1 barcode is selectively enriched in BRD4-overexpressing HeLa droplets.
• Scalable to large DELs (96×96×96 combinatorial space across three scaffolds) with nanopore sequencing–based enrichment quantification.

Conclusion

The platform bridges functional intracellular DEL screening with high spatial fidelity and quantitative readouts—enabling both PCR- and sequencing-based hit identification while preserving native biomolecular context.