DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information

Abstract

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.

Keywords: DNA-encoded chemical libraries; combinatorial chemistry; drug discovery.

Figure 1

Schematic representation of antibody phage display libraries and of DNA-encoded chemical libraries. In antibody phage display libraries, an antibody fragment (e.g., a scFv fragment) is displayed on the surface of filamentous phage and could potentially bind to a cognate protein target. The genetic information coding for the recombinant antibody is contained in the genome of the bacteriophage. In full analogy, small organic molecules can be attached to DNA-fragments, serving as amplifiable identification barcodes.

Figure 2

Schematic representation of biopanning procedures with antibody phage display libraries and with DNA-encoded chemical libraries. In the first case, a large library of phage antibodies is incubated with the target protein of interest, immobilized on a solid support. Selective binders are captured on the affinity support, while the majority of other phage antibodies (which do not bind toia the target) can be washed away. Selected phage particles can be amplified by infecting bacteria, leading to an amplification step and to the generation of more phage particles, which can be submitted to a second round of panning. Alternatively, infected bacteria can be plated onto selective plates and individual colonies correspond to distinct monoclonal antibody clones. Similarly, large collections of organic molecules (individually tagged with DNA barcodes) can be interrogated using an affinity capture procedure. Preferentially enriched molecules can be identified by PCR amplification of the DNA tags, followed by high-throughput DNA sequencing.

Figure 3

Schematic representation of “single-pharmacophore” and “dual-pharmacophore” DNA-encoded chemical libraries. Next to the double-helix representation of the DNA structure, a linear schematic representation is also displayed, as this graphical representation is used in subsequent slides, describing encoding procedures.

Figure 4

Schematic representation of encoding strategies for DNA-recorded chemical libraries. (a) In the simplest implementation of this technology, each building block in the synthesis procedure is encoded (i.e., identified) by a distinctive double-stranded DNA fragment. After each chemical reaction, the identity of the newly introduced building block is provided by an additional DNA fragment, which is ligated to the nascent DNA structure. (b) In a variation of the procedure described before, the complementary DNA strands are connected by a linker, which also supports the growth of the nascent molecule. (c) In a different encoding procedure, a first building block is attached at the 5’ end of an oligonucleotide, which contains a suitable identification code. After a second building block has been added to the nascent molecule by chemical reaction, its identity is encoded by the annealing of a partially complementary oligonucleotide, followed by a Klenow polymerization procedure.

Figure 5

Schematic representation of encoding strategies for DNA-templated synthesis of chemical libraries. (a) An oligonucleotide template (here depicted for the construction of a molecule based on two building blocks) is annealed with a chemically modified oligonucleotide, which transfers a chemical moiety to the nascent molecule. At the end of the coupling step, the cleavable connection between the building block B and the corresponding oligonucleotide (depicted with a dotted line). (b) In a modified procedure, a general template (containing poly-I stretches for annealing with various coding segments) is sequentially hybridized with oligonucleotide derivatives, which transfer building blocks onto a nascent molecule. (c) In YoctoReactor™ technology, hairpin structures (containing a coding sequence and a building block, are used to mediate successive cycles of DNA ligation and of chemical reactions. Cleavable linkers are indicated with a dotted line. (d) In DNA routing strategies, long oligonucleotides (containing multiple coding sequences) are sequentially hybridized to columns carrying complementary oligonucleotides. Individual column hybridization steps dictate which chemical reaction (i.e., which building block addition) is performed on a given nascent molecule.

Figure 6

Encoding strategies for dual-pharmacophore encoded chemical libraries. (a) General structure of ESAC library members, which are decoded by hybridization onto oligonucleotide microarrays. (b) In a more powerful procedure, ESAC library members are encoded with a methodology, that leads to the simultaneous presence of two codes (identifying building blocks A and B) onto one of the two complementary strands. (c) Encoding strategy, compatible with high-throughput sequencing decoding procedures. Members of the “red” sublibrary are constructed by the coupling of building blocks to a general oligonucleotide carrying an abasic site, followed by encoding by ligation assisted by a splint degradable oligo. By contrast, members of the “blue” sublibrary are created by coupling building blocks to the 5’ extremity of suitable oligonucleotides, carrying an identification code. The two sublibraries can then be annealed followed by a Klenow polymerization step. (c) In one of the possible implementations of the technology, chemically-modified PNAs are hybridized to DNA templates, which carry suitable complementary coding sequences.

Figure 7

Selection methods for DNA-encoded chemical libraries. (a) Library members are captured on a target protein, immobilized on a solid support. (b) DNA derivatives, capable of binding to a target protein with sufficient stability in given experimental conditions, are separated from non-binding library members by chromatography or by electrophoresis. (c) Library members encoded by single-stranded DNA molecules, capable of binding to a target protein of interest equipped with a suitable oligonucleotide primer, can be identified by a DNA polymerization step, followed by PCR amplification. (d) Capture of preferentially-binding library members by photo-crosslinking.

Figure 8

Illustrative fingerprints of selection results, performed with a library consisting of two (a) or three (b) sets of building blocks.

Figure 9

Selected examples of binding molecules, isolated from DNA-encoded chemical libraries. The individual examples show the general library designs, which were used for the selections. The color-code of the building blocks is retained when displaying the chemical structure of the hit molecule, facilitating the detection of synthetic strategies and of modular structures.

Figure 9

Selected examples of binding molecules, isolated from DNA-encoded chemical libraries. The individual examples show the general library designs, which were used for the selections. The color-code of the building blocks is retained when displaying the chemical structure of the hit molecule, facilitating the detection of synthetic strategies and of modular structures.

Figure 9

Selected examples of binding molecules, isolated from DNA-encoded chemical libraries. The individual examples show the general library designs, which were used for the selections. The color-code of the building blocks is retained when displaying the chemical structure of the hit molecule, facilitating the detection of synthetic strategies and of modular structures.

Figure 10

Methods for the fluorescent labeling of library members, enabling fluorescence polatization measurement procedures. For single-pharmacophore library members, hits can be resynthesized as fluorescently-labeled compounds, or on oligonucleotides (DNA or LNA) which carry the fluorophore on the same strand or on a complementary strand. Similarly, hits from dual-pharmacophore chemical libraries can be re-synthesized as fluorescent molecules (with a suitable linker connecting the two building blocks) or on complementary oligonucleotides (one of which is fluorescently labeled).