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<p>DNA-encoded chemical libraries (DELs) are collections of DNA-linked small molecules, where each synthetic small molecule is covalently attached to a unique DNA barcode that encodes its identity. This technology harnesses the power of organic chemistry and genetics, which extends the application of molecular evolution and natural selection to the discovery of specific small molecules binders to protein targets of interest. Rather than discretely screening individual molecules, up to billions of DNA-encoded small molecules can be assessed collectively by a selection assay in a single tube. As a result, the high sensitivity, low cost, and unprecedented level of molecular complexity of DELs allow rapid generation of novel bioactive compounds. While powerful, this approach has its own limitations, including limited target scope and selection strategies. Currently, DEL targets have been largely limited to biochemically purified proteins and used in affinity-based selections assays. In the first area of this work, we address both these limitations by capitalizing on the power of affinity labeling. This allows DELs to be applied to protein targets within and on living cells and expands the power of DNA-encoding to the identification of small molecules with specific biological functions beyond binding. </p>
<p>In the second area, we harnessed affinity labeling and DNA sequence analysis to develop multiplexed small molecule ligand binding assays. This method is the initial demonstration of split-and-pool ligand binding assays using DNA-linked small molecule probes. We used this approach in a high-throughput screening campaign to identify selective inhibitors by screening 1000 compounds against 5 bromodomain proteins concurrently. In addition, this approach was utilized to rank order the affinity of a 96-member library of DNA-linked ligands to a protein simultaneously, which significantly increases the throughput of ligand binding assays while keeps the cost low. </p>
<p>Lastly, we developed proximity-induced selection assays to enrich ligands from DELs. This approach involves uncaging or installation of a biotin purification tag on the DNA construct either through photo-deprotection of a protected biotin group using a light emitting protein tag or by amine acylation using an engineered biotin ligase. Compared to affinity labeling-based selection approaches, this approach results in improved recovery of ligands and, at the same time, removes the onerous requirement of protein purification. The enzyme-mediated proximity labeling approach should serve as a convenient tool for molecular discovery with DELs. </p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/19949822 |
| Date | 01 June 2022 |
| Creators | Bo Cai (12708119) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Applications_of_Affinity_Labeling_with_DNA-Encoded_Chemical_Libraries/19949822 |
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