Anjali Dixit & Brian M. Paegel

Nature Protocols (2025)

DOI: 10.1038/s41596-025-01190-4

Abstract

This protocol details the construction of solid-phase DNA-encoded libraries (DELs) that merge one-bead–one-compound (OBOC) combinatorial chemistry with high-throughput DNA sequencing. Photocleavable linkers allow library members to be released from individual polystyrene beads while the encoding DNA tag remains attached, enabling both affinity and activity-based screening. Step-by-step instructions cover bifunctional linker synthesis, split-and-pool combinatorial cycles, enzymatic DNA ligation, quality control by single-bead qPCR/NGS, and validation by MALDI-TOF MS. The entire workflow is completed in ~4 weeks without specialized automation and furnishes 10⁴–10⁶ photocleavable, off-DNA-screenable compounds ready for microfluidic or FACS-based functional assays.

Summary

Solid-phase DEL technology overcomes the limitations of conventional on-DNA DELs by physically isolating each library member on a bead and permitting photochemical release for cell-free or cell-based activity assays. The protocol begins with synthesis of a dual-scale bead set (160 µm QC beads and 10 µm screening beads) displaying a coumarin/arginine-modified linker terminated with a propargylglycine alkyne and a photocleavable o-nitrobenzyl bromide handle. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) installs an azide-modified hairpiece DNA (N₃-HDNA) to create bifunctional beads. Combinatorial three-cycle chemistry (diamine displacement → Fmoc-amino acid acylation → carboxylic acid acylation) is performed in 96-well filter plates with alternating aqueous DNA-ligation steps that append unique oligonucleotide codes for every building block (BB). A final bead-specific barcode (BSB) ligation increases sequence diversity to >3,000 unique codes per compound. Quality control is rigorous: every cycle is monitored by qPCR quantification of DNA molecules per bead (MPB), LC–MS of TFA-cleaved QC beads, and single-bead Sanger sequencing matched to MALDI-TOF MS. Baseline NGS of 1,500-bead aliquots establishes BB frequency thresholds for hit calling. A 300-mg synthesis routinely yields 6 × 10⁸ beads, equivalent to ~65,000 copies of each compound in a 9,216-member two-cycle library—sufficient for >2,000 microfluidic droplet screens or 50 min FACS analysis. Data analysis scripts (Python/Figshare) decode reads via UMI aggregation and Hamming-distance error correction, enabling structure–activity relationship (SAR) extraction and machine-learning training sets.

Highlights

1. Photocleavable, off-DNA screening: Library members are released from beads under UV light while DNA tags remain attached, enabling dose–response and cellular phenotypic assays impossible with conventional on-DNA DELs. 2. One-bead–one-compound plus DNA encoding: Combines OBOC spatial isolation with massively parallel sequence deconvolution, eliminating tedious hit resynthesis and providing direct SAR data. 3. Modular split-and-pool chemistry: Three-cycle protocol (amine→amide→amide) uses 96-well plates and standard peptide-coupling reagents; readily reconfigurable to other medicinally relevant reactions validated by DNA-encoded reaction rehearsal. 4. Built-in quality control: Every synthetic and enzymatic step is tracked by single-bead qPCR, PAGE, LC–MS, and MALDI-TOF MS; ≥90 % of QC beads must yield correct m/z and clean Sanger reads for library release. 5. Scalable & accessible: All reagents are commercially available; no automation required; 4-week timeline from bead swelling to NGS-validated library; yields enough material for thousands of screens at 10–30 compound equivalents per assay.

Conclusion

The solid-phase DEL protocol described here delivers a versatile, quality-controlled route to 10⁴–10⁶ photocleavable small molecules that can be interrogated in both biochemical and cell-based assays. By integrating OBOC spatial segregation with DNA encoding, researchers gain the throughput advantages of DEL selection while preserving the pharmacological fidelity of off-DNA compound evaluation. Rigorous QC checkpoints at every cycle ensure that libraries entering screening are chemically and physically intact, minimizing false positives and maximizing SAR information richness. The workflow is intentionally manual and low-cost, making functional DEL screening accessible to any laboratory equipped for routine solid-phase peptide synthesis and qPCR.