Nonlinear manipulation and analysis of large DNA datasets.

Information processing functions are essential for organisms to perceive and react to their complex environment, and for humans to analyze and rationalize them. While our brain is extraordinary at processing complex information, winner-take-all, as a type of biased competition is one of the simplest models of lateral inhibition and competition among biological neurons. It has been implemented as DNA-based neural networks, for example, to mimic pattern recognition. However, the utility of DNA-based computation in information processing for real biotechnological applications remains to be demonstrated. In this paper, a biased competition method for nonlinear manipulation and analysis of mixtures of DNA sequences was developed. Unlike conventional biological experiments, selected species were not directly subjected to analysis. Instead, parallel computation among a myriad of different DNA sequences was carried out to reduce the information entropy. The method could be used for various oligonucleotide-encoded libraries, as we have demonstrated its application in decoding and data analysis for selection experiments with DNA-encoded chemical libraries against protein targets.

Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase IX (CA IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions.

High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries.

DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG.

Drug discovery with DNA-encoded chemical libraries.

DNA-encoded chemical libraries represent a novel avenue for the facile discovery of small molecule ligands against target proteins of biological or pharmaceutical importance. Library members consist of small molecules covalently attached to unique DNA fragments that serve as amplifiable identification barcodes. This encoding allows the in vitro selection of ligands at subpicomolar concentrations from large library populations by affinity capture on a target protein of interest, in analogy to established technologies for the selection of binding polypeptides (e.g., antibodies). Different library formats have been explored by various groups, allowing the construction of chemical libraries comprising up to millions of DNA-encoded compounds. Libraries before and after selection have been characterized by PCR amplification of the DNA codes and subsequent relative quantification of library members using high-throughput sequencing. The most enriched compounds have then been further analyzed in biological assays, in the presence or in the absence of linked DNA. This article reviews experimental strategies used for the construction of DNA-encoded chemical libraries, revealing how selection, decoding, and hit validation technologies have been used for drug discovery programs.

Isolation of potent and specific trypsin inhibitors from a DNA-encoded chemical library.

Collections of chemical compounds, individually attached to unique DNA fragments serving as amplifiable identification bar codes, are generally referred to as "DNA-encoded chemical libraries". Such libraries can be used for the de novo isolation of binding molecules against target proteins of interest. Here, we describe the synthesis and use of a DNA-encoded library based on benzamidine analogues, which allowed the isolation of a trypsin inhibitor with an IC(50) value of 3.0 nM, thus representing a >10 000-fold potency improvement compared to the parental compound. The novel trypsin inhibitor displayed an excellent selectivity toward other serine proteases. This study indicates that DNA-encoded libraries can be used for the facile "affinity maturation" of suboptimal binding compounds, thus facilitating drug development.

High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries.

DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.

Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions.

DNA-encoded chemical libraries are increasingly being employed for the identification of binding molecules to protein targets of pharmaceutical relevance. Here, we describe the synthesis and characterization of a DNA-encoded chemical library, consisting of 4000 compounds generated by Diels-Alder cycloaddition reactions. The compounds were encoded with unique DNA fragments which were generated through a stepwise assembly process and serve as amplifiable bar codes for the identification and relative quantification of library members.

20 years of DNA-encoded chemical libraries.

The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.

Isolation of a small-molecule inhibitor of the antiapoptotic protein Bcl-xL from a DNA-encoded chemical library.

Bcl-xL is an antiapoptotic member of the Bcl-2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl-xL from a DNA-encoded chemical library using affinity-capture selections and massively parallel high-throughput sequencing of >30,000 sequence tags of library members. The most potent binder identified, compound 19/93 [(R)-3-(amido indomethacin)-4-(naphthalen-1-yl)butanoic acid], bound to Bcl-xL with a dissociation constant (K(d)) of 930 nM and was able to compete with a Bak-derived BH3 peptide, an antagonist of Bcl-xL function.

DNA-encoded chemical libraries for the discovery of MMP-3 inhibitors.

Encoded self-assembling chemical (ESAC) libraries are characterized by the covalent display of chemical moieties at the extremity of self-assembling oligonucleotides carrying a unique DNA sequence for the identification of the corresponding chemical moiety. We have used ESAC library technology in a two-step selection procedure for the identification of novel inhibitors of stromelysin-1 (MMP-3), a matrix metalloproteinase involved in both physiological and pathological tissue remodeling processes, yielding novel inhibitors with micromolar potency.