Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

On-DNA Palladium-Catalyzed Hydrogenation-like Reaction Suitable for DNA-Encoded Library Synthesis.

Herein we describe a method to orthogonally remove on-DNA N-Cbz, N-Alloc, N-Allyl, O-Bn, and O-Allyl protecting groups in the presence of other common protecting groups to afford free amines and carboxylic acids, respectively. The developed method uses NaBH4 as the source of hydrogen in the presence of Pd(OAc)2 under DNA aqueous conditions. In addition, under the developed conditions we were able to successfully hydrogenate triple and double bonds to totally saturated compounds. Furthermore, we introduce a new alternative procedure to reduce azides and aromatic nitro compounds to primary amines.

Normalization of DNA encoded library affinity selection results driven by high throughput sequencing and HPLC purification.

The output of an affinity selection screening results in a huge amount of valuable data that, after conducting the appropriate analysis, lead to the correct identification of the compounds enriched in the target of interest. The approach chosen to perform these analyses has become a key step in the development of a successful DNA Encoded Library platform. In this paper, we describe the combination of High Performance Liquid Chromatography purification during the library production with the Next Generation Sequencing analysis of the libraries to assess the yield of the chemical reactions prior to the affinity selection. This process allows us, apart from achieving higher quality libraries, to enable a normalization analysis of the affinity selection output, thus minimizing the bias induced by the chemical yield of each reaction as a misleading factor within the analysis and subsequent compound short-listing for off-DNA synthesis.

High-Throughput DNA-Encoded Libraries Affinity Selection Platform for Binder Identification with Solid Support Protein Immobilization.

DNA-encoded libraries (DELs) have demonstrated to be one of the most powerful technologies within the ligand identification toolbox, widely used either in academia or biotech and pharma companies. DEL methodology utilizes affinity selection (AS) as the approach to interrogate the protein of interest for the identification of binders. Here we present a high-throughput, fully automated AS platform developed to fulfill industrial standards and compatible with different assay formats to improve the reproducibility of the AS process for DEL binders identification. This platform is flexible enough to virtually set aside all kinds of DELs and AS methods and conditions using immobilized proteins. It bears the two main immobilization methods to support of the proteins of interest: magnetic beads or resin tip columns. A combination of a broad variety of protocol options with a wide range of different experimental conditions can be set up with a throughput of 96 samples at the same time. In addition, small modifications of the protocols provide the platform with the versatility to run not only the routine DEL screens, but also test covalent libraries, the successful immobilization of the proteins of interest, and many other experiments that may be required. This versatile AS platform for DEL can be a powerful instrument for direct application of the technology in academic and industry settings.

Occurrence and diversity of the oxidative hydroxyhydroquinone pathway for the anaerobic degradation of aromatic compounds in nitrate-reducing bacteria.

The nitrate-reducing betaproteobacteria Azoarcus anaerobius and Thauera aromatica AR-1 use an oxidative mechanism to anaerobically degrade resorcinol and 3,5-dihydroxybenzoate (3,5-DHB), respectively, rendering hydroxyhydroquinone as intermediate. The first pathway step is performed by a dimethylsulphoxide-reductase family hydroxylase. The gene cluster coding for the pathway is homologous in these strains. Only these two Rhodocyclales are known to follow this anaerobic pathway, and nothing is known about its distribution in prokaryotes. To determine the relevance and diversity of this strategy in nature, we enriched for bacteria able to oxidize resorcinol or 3,5-DHB under denitrifying conditions. Nitrate-reducing bacteria able to degrade these compounds were present in soil, aquifer and marine sediments. We were able to isolate a number of strains with this capacity from soil and aquifer samples. Amplicon libraries of rehL, the gene encoding the first step of this pathway, showed an overall low diversity, most sequences clustering with either pathway enzyme. Isolates belonging to the Beta- and Gammaproteobacteria able to grow on these substrates revealed rehL homologues only in strains belonging to Thauera and Azoarcus. Analysis of sequenced genomes in the databases detected the presence of highly similar clusters in two additional betaproteobacteria and in the gammaproteobacterium Sedimenticola selenatireducens, although anaerobic growth on a dihydroxyaromatic could only be confirmed in Thauera chlorobenzoica 3CB-1. The presence of mobile elements in the flanking sequences of some of the clusters suggested events of horizontal gene transfer, probably contributing to expand the pathway to a broader host range within the Proteobacteria.

tagFinder: A Novel Tag Analysis Methodology That Enables Detection of Molecules from DNA-Encoded Chemical Libraries.

Available tools to analyze sequencing data coming from DNA-encoded chemical libraries (DELs) are often limited to in-house methods, which usually rely on strictly looking for the particular DEL structure used. Current methods do not take into account technological errors, such as library codification and sequencing errors, when detecting the sequences. The vast amount of data produced by next-generation sequencing of DEL screens is usually enough to extract the minimum information needed for compound identification. Here, we report a methodology to deconvolute encoding oligonucleotides, thus optimizing the sequencing power regardless of the library size, design complexity, or sequencing technology chosen. tagFinder is a highly flexible tool for fast tag detection and thorough DEL results characterization, which requires minimal hardware resources, scales linearly, and does not introduce any analytical error. The methodology can even deal with sequencing errors and PCR duplicates on single- or double-stranded DNA, enhancing the analytical detection and quantification of molecules and the informativeness of the entire process. Source code is available at https://github.com/jamigo/tagFinder .