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Solid-phase DNA-encoded library synthesis: a master builder’s instructions

Nature Protocols (2025)Cite this article

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Abstract

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.

Key points

  • This solid-phase DNA-encoded library synthesis protocol uses combinatorial split-and-pool synthesis to generate diverse, photocleavable chemical compounds encoded by DNA tags.

  • Solid-phase chemistry using a photocleavable linker allows off-DNA release and evaluation of small-molecule library members, enabling activity-based screening for DNA-encoded libraries.

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Fig. 1: Schematic of OBOC-DEL synthesis.
Fig. 2: Library linker synthesis.
Fig. 3: BB QC flowchart.
Fig. 4: N3-HDNA preparation and characterization.
Fig. 5: Example encoding formats using a six-position encoding scheme.
Fig. 6: Example PAGE analysis and successful single-bead deconvolution by Sanger DNA sequencing.
Fig. 7: NGS data analysis workflow.

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Data availability

Example NGS decoding scripts and raw data files accompanying figures within this manuscript can be found at Example NGS Decoding script via Figshare at https://figshare.com/s/425bb12ef2b5f20bb856 (ref. 45). The raw data files can be found via Figshare at https://figshare.com/s/5453000e506029b5298c (ref. 46). Source data are provided with this paper.

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Acknowledgements

This work was supported by a grant award from the National Institutes of Health (grant no. GM140890) to B.M.P.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA

    Anjali Dixit & Brian M. Paegel

  2. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    Brian M. Paegel

  3. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA

    Brian M. Paegel

Authors
  1. Anjali Dixit
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  2. Brian M. Paegel
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Contributions

A.D. collected and analyzed data and prepared a preliminary draft of the manuscript with guidance from B.M.P. All authors read, edited and approved the final manuscript.

Corresponding author

Correspondence to Brian M. Paegel.

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Nature Protocols thanks Xiaojie Lu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Barhoosh, H. et al. ACS Cent Sci. 10, 1960–1968 (2024): https://doi.org/10.1021/acscentsci.4c01218

Fitzgerald, P. et al. ACS Med. Chem. Lett. 14, 1295–1303 (2023): https://doi.org/10.1021/acsmedchemlett.3c00159

Hackler, A. et al. ACS Comb Sci. 22, 25–34 (2020): https://doi.org/10.1021/acscombsci.9b00153

Supplementary information

Supplementary Information

Supplementary Tables 1–6 and Figs. 1–7.

Source data

Source Data Fig. 4

Source plots for chromatography plots and mass spectral data.

Source Data Fig. 6

Unprocessed PAGE gels and source MALDI-TOF MS data for figure. Available in Supplementary Information as well.

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Dixit, A., Paegel, B.M. Solid-phase DNA-encoded library synthesis: a master builder’s instructions. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01190-4

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