DNA-encoded chemical library

DNA-encoded chemical libraries (DECL) is a technology for the synthesis and screening on an unprecedented scale of collections of small molecule compounds.

The technique enables the mass creation and interrogation of libraries via affinity selection, typically on an immobilized protein target.

A homogeneous method for screening DNA-encoded libraries (DELs) has recently been developed which uses water-in-oil emulsion technology to isolate, count and identify individual ligand-target complexes in a single-tube approach.

(Eötvös Loránd University Budapest Hungary) in 1982, and described it including the method of synthesis of combinatorial libraries and that of a deconvolution strategy in a document notarized in the same year.

[5] The concept of DNA-encoding was first described in a theoretical paper by Sydney Brenner and Richard Lerner in 1992 in which was proposed to link each molecule of a chemically synthesized entity to a particular oligonucleotide sequence constructed in parallel and to use this encoding genetic tag to identify and enrich active compounds.

[7][8] Brenner and Janda suggested to generate individual encoded library members by an alternating parallel combinatorial synthesis of the heteropolymeric chemical compound and the appropriate oligonucleotide sequence on the same bead in a “split-&-pool”-based fashion (see below).

), mainly to avoid inconvenient solid phase DNA synthesis and to create easily screenable combinatorial libraries in high-throughput fashion.

ESAC technology sets itself apart being a combinatorial self-assembling approach which resembles fragment based hit discovery (Fig 1b).

Here DNA annealing enables discrete building block combinations to be sampled, but no chemical reaction takes place between them.

[13] This new approach helps to increase practically unlimitedly the number of components of DNA encoded combinatorial libraries (DECLs).

Similarly, diene carboxylic acids used as scaffolds for library construction at the 5’-end of amino modified oligonucleotide, could be subjected to a Diels-Alder reaction with a variety of maleimide derivatives.

The synthetic and encoding strategies described above enable the facile construction of DNA-encoded libraries of a size up to 104 member compounds carrying two sets of “building blocks”.

However the stepwise addition of at least three independent sets of chemical moieties to a tri-functional core building block for the construction and encoding of a very large DNA-encoded library (comprising up to 106 compounds) can also be envisaged.

[18] Preferential binders isolated from an affinity-based selection can be PCR-amplified and decoded on complementary oligonucleotide microarrays[19] or by concatenation of the codes, subcloning and sequencing.

The compound represents the core structure of a series of portable albumin binding molecules and of Albufluor a recently developed fluorescein angiographic contrast agent currently under clinical evaluation.

[22] The design of Halpin and Harbury enabled alternating rounds of selection, PCR amplification and diversification with small organic molecules, in complete analogy to phage display technology.

The DNA-routing machinery consists of a series of connected columns bearing resin-bound anticodons, which could sequence-specifically separate a population of DNA-templates into spatially distinct locations by hybridization.

[23] In 2001 David Liu and co-workers showed that complementary DNA oligonucleotides can be used to assist certain synthetic reactions, which do not efficiently take place in solution at low concentration.

[26] Using a DNA-templated set-up and sequence-programmed synthesis Liu and co-workers generated a 64-member compound DNA encoded library of macrocycles.

Chemical reactions are performed via a stepwise procedure and after each step the DNA is ligated and the product purified by polyacrylamide gel electrophoresis.

Cleavable linkers (BB-DNA) are used for all but one position yielding a library of small molecules with a single covalent link to the DNA code.

Furthermore, the intimate connection between the code and the BB on the oligo-BB moieties which are mixed combinatorially in a single pot confers a high fidelity to the encoding of the library.

A homogeneous method for screening yoctoreactor libraries (yR) has recently been developed which uses water-in-oil emulsion technology to isolate individual ligand-target complexes.

Biologically active hits are identified in a single round of BTE characterized by a low false positive rate.

Following selection from DNA-encoded chemical libraries, the decoding strategy for the fast and efficient identification of the specific binding compounds is crucial for the further development of the DEL technology.

[18] After selection and PCR amplification of the DNA-tags of the library compounds, concatamers containing multiple coding sequences were generated and ligated into a vector.

Following Sanger sequencing of a representative number of the resulting colonies revealed the frequencies of the codes present in the DNA-encoded library sample before and after selection.

It consists of an arrayed series of microscopic spots (‘features’ or ‘locations’) containing few picomoles of oligonucleotides carrying a specific DNA sequence.

Subsequently, the oligonucleotide tags of the binding compounds isolated from the selection are PCR amplified using a fluorescent primer and hybridized onto the DNA-microarray slide.

**Fig. 1** **DNA-encoded library displaying chemical compounds** Schematic representation of DNA-encoded library displaying chemical compounds directly attached to oligonucleotides. a) Library generated by “stepwise combinatorial” assembling presenting a single oligonucleotide covalently linked to a putative binding molecule. b) Library construct in “combinatorial self-assembling” fashion ( E ncoded S elf- A ssembling C hemical library). Multiple pairing oligonucleotides display a covalently linked binding molecule

**Fig. 3** **DNA-encoded library by "Split-&-Pool stepwise coupling of coding DNA fragments to nascent organic molecules** An initial set of multifunctional building blocks (FGn represents the different orthogonal functional groups) are covalently conjugated to a corresponding encoding oligonucleotide and reacted in a split-&-pool fashion on a specific functional group (FG1 in red) with a suitable collection of reagents. Following enzymatic encoding, a further round of split-&-pool is initiated. At this stage the second functional group (FG2 in blue) undergoes an additional reaction step with a different set of suitable reagents. The identity of the final modification could be ensured yet again by enzymatic DNA encoding by means of a further oligonucleotide carrying a specific coding region.

**Fig. 4** **ESAC library technology overview** Small organic molecules are coupled to 5’-amino modified oligonucleotides, containing a hybridization domain and a unique coding sequence, which ensure the identity of the coupled molecule. The ESAC library can be used in single pharmacophore format (a), in affinity maturations of known binders (b), or in de novo selections of binding molecules by self assembling of sublibraries in DNA-double strand format (c) as well as in DNA-triplexes (d). The ESAC library in the selected format is used in a selection and read-out procedure (e). Following incubation of the library (i) with the target protein of choice (ii) and washing of unbound molecules (iii), the oligonucleotide codes of the binding compounds are PCR-amplified and compared with the library without selection on oligonucleotide micro-arrays (iv, v). Identified binders/binding pairs are validated after conjugation (if appropriate) to suitable scaffolds (vi).

**Fig. 2** **DNA-encoded library by ‘DNA-templated synthesis’** A library of oligonucleotides (i.e. 64 different oligonucleotides) containing three coding regions was hybridized to a library of reagent compound-oligonucleotide conjugates (i.e. 4 reagent oligonucleotide conjugates), able of pairing with the initial coding domain of the template oligonucleotide. After transferring of the compounds on the corresponding oligonucleotide template, the synthesis cycle was repeated the desired number of times with further sets of carrier compound-oligonucleotide conjugates (i.e. two rounds with four carrier compound-oligonucleotide conjugates per round). Subsequently, functional selection was performed and the sequence of the binding template amplified by PCR. Thus, DNA-sequencing allowed the identification of the binding molecule.

**Fig. 6** **YoctoReactor library assembly.** Stepwise assembly of a DEL library using YoctoReactor technology. A 3-way reactor is shown here. (a) Position 1 (P1) and P2 BB are brought into proximity and undergo a chemical reaction in the presence of a helper oligonucleotide in P3. (b) The structure is purified by polyacrylamide gel electrophoresis (PAGE), the P1 and P2 DNA is ligated and the P2 linker is cleaved. (c) P3 BB is annealed to the P1-P2 ligation product from step b, and a chemical reaction between P2 and P3 BBs takes place. (d) The reaction product is purified by PAGE, the DNA is ligated and P3 linker is cleaved yielding a compound (OOO) covalently attached to the folded yR. (e) The yR is dismantled by primer extension yielding a double-stranded display product exposing the reaction product for selection and molecular evolution.