Advancing DNA-Encoded Library Technology Through Enzyme-Assisted Synthesis and Encoded Linker Selections
EMBARGOED UNTIL 2026-12-16
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2025
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Doctoral Thesis
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EMBARGOED UNTIL 2026-12-16
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Abstract
Within pharmaceutical research, both in industry and academia, DNA-encoded chemical library (DEL) technology is by now an established method allowing the rapid identification of ligands against a multitude of biologically relevant targets. By encoding the synthetic history and identity of small molecules within the sequence of a covalently attached DNA tag, DELs can be synthesised, selected and stored as mixtures of molecules. Substantial DELs can be rapidly assembled through combinatorial synthesis; however, library quality is tributary to the availability of DNA-compatible synthetic methods which ensure uniformly high conversions over a wide range of building blocks. This is particularly true for advanced stages of library construction when the purification of individual compounds is impossible and gets exacerbated with the inclusion of more diversity elements.
In this thesis, two approaches were examined to advance DEL technology by improving library quality. In the first project, the application of biocatalytic methods for DEL synthesis was explored. While mild reaction conditions and high selectivity make enzymatic catalysis inherently interesting for on-DNA reactions, this synthetic modality has so far been largely unexplored due to significant implementation challenges. A cascade of CoA-ligases and N-acyltransferases was applied for amide-bond formations on DNA-tagged compounds and proved to preserve DNA tag integrity. The identified wild-type enzymes were used as starting points for enzyme engineering, enabling improved conversions and effectively increasing the functional substrate scope. To further assess the utility of the developed cascade for DEL construction, the enzymes were applied for the late-stage functionalisation of DNA-tagged molecules. The N-acyltransferases were found to be remarkably accepting of a wide range of substrates, including very bulky molecules, and allowed for average conversions above 91 % for tested aromatic carboxylic acids and DNA-tagged amines.
The development of more DNA-compatible synthetic methods for small molecule construction is one approach to increase library quality. Dual-display technology represents an alternative strategy, allowing the construction of vast DELs through the combinatorial hybridisation of smaller sub-libraries of higher purity without compromising on library quality. However, the identification of adequate linker structures to join identified ligand pairs into one heterobifunctional molecule remains a challenge, not only in dual-display DEL technology, but also in fragment-based drug discovery. Indeed, the fragment linking step often demands substantial synthetic effort, typically limiting the number of structures that can be explored. In the second project presented in this thesis, a novel strategy for rapidly exploring linker structures by enabling encoded linker selections was developed. A poised library comprising 141 diverse linker structures was constructed. The resulting library was used for rapidly elaborating two fragment display libraries by conjugating known ligands to validated control proteins. Affinity-based selections were performed with the obtained libraries and allowed the successful validation of the developed setup. Enrichment of conjugates was observed not only due to avidity and rebinding effects but also for individual linker structures, demonstrating the potential of the approach for preferentially enriching specific molecules and therefore identifying optimal linkages between fragment pairs.
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ETH Zurich
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DNA-encoded chemical libraries
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08641 - Scheuermann, Jörg (Tit.-Prof.) / Scheuermann, Jörg (Tit.-Prof.)
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CRSII5_198673 / 1 - Enzymatic Construction of DNA-Encoded Chemical Libraries (EnzyDEL) (SNF)