title: Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.
abstract: DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis.
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zürich, Switzerland.
authors.forename: Gunther
authors.initials: G
authors.lastname: Zimmermann
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zürich, Switzerland.
authors.forename: Yizhou
authors.initials: Y
authors.lastname: Li
authors.affiliation: Philochem AG, Libernstrasse 3, 8112, Otelfingen, Switzerland.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Rieder
authors.affiliation: Philochem AG, Libernstrasse 3, 8112, Otelfingen, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Mattarella
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zürich, Switzerland.
authors.forename: Dario
authors.initials: D
authors.lastname: Neri
authors.affiliation: Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, ETH Zürich, Vladimir-Prelog-Weg 3, 8093, Zürich, Switzerland.
authors.forename: Jörg
authors.initials: J
authors.lastname: Scheuermann
mesh_terms: D000086:Acetazolamide / Q000737:chemistry / Q000378:metabolism; D000071231:Carbonic Anhydrase IX / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D055808:Drug Discovery; D005456:Fluorescent Dyes / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing; D008024:Ligands; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D054852:Small Molecule Libraries / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D009841:Oligonucleotides; D054852:Small Molecule Libraries; C477371:locked nucleic acid; D004247:DNA; D000071231:Carbonic Anhydrase IX; D000086:Acetazolamide
keywords: DNA-encoded chemical libraries; drug discovery; fluorescence anisotropy; high-throughput screening	; hit validation; koff