DNA Encoded Chemical Libraries (DEL) are a new technology for the synthesis and screening of collections of chemical compounds of unprecedented size and quality.

The DNA Encoded Chemical Library technology has been recently developed to improve the drug discovery process. In DEL chemical compounds are individually conjugated to short DNA fragments that serve as identification bar codes. In contrast to conventional screening procedures such as high-throughput screening, biochemical assays are not required for binder identification, in principle allowing the isolation of binders to a wide range of proteins historically difficult to tackle with conventional screening technologies. The availability of binders to such pharmacologically important, but so-far “undruggable” target proteins promises to facilitate the development of new generations of drugs for diseases that could not be treated so far and in general the discovery of target specific molecular compounds.

[edit] Phage display and DNA Encoded Chemical libraries

DNA encoded chemical libraries bear resemblance to the antibody phage display technology. In antibody phage display, antibodies are physically linked to phage particles that bear the gene coding for the attached antibody, which is equivalent to a physical linkage of a “phenotype” (the protein) and a “genotype” (the gene encoding for the protein).^[1] In DEL the linkage of a molecule to an identifier DNA tag allows the facile identification of binding molecules. After affinity capture procedures on an immobilized target protein of choice, the DNA-tags of the binding molecules are amplified by polymerase chain reaction (PCR) and subsequently identified (e.g. by DNA sequencing). By means of PCR even single molecules DNA-tagged can be amplified and identified.

[edit] History

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 (Encoded Self-Assembling Chemical library). Multiple pairing oligonucleotides display a covalently linked binding molecule

The concept of DNA-encoding was first described in a theoretical paper by Brenner and 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.^[2] In 1993 the first practical implementation of this approach was presented by S. Brenner and K. Janda and similarly by the group of M.A. Gallop.^[3][4] 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).^[3]

Since unprotected DNA is restricted to a narrow window of conventional reaction conditions, until the end of 1990s a number of alternative encoding strategies were envisaged (i.e. MS-based compound tagging, peptide encoding, haloaromatic tagging, encoding by secondary amines, semiconductor devices.), mainly to avoid inconvenient solid phase DNA synthesis and to create easily screenable combinatorial libraries in high-throughput fashion.^[5] However, the selective amplifiability of DNA greatly facilitates library screening and it becomes indispensable for the encoding of organic compounds libraries of this unprecedented size. Consequently, at the beginning of 2000s DNA-combinatorial chemistry experienced a revival.

Several groups at the beginning of 2000s further developed the technology. The resulting libraries can be grouped in libraries generated by “stepwise combinatorial” and library construct in “combinatorial self-assembling” fashion (see below and Fig.1).

[edit] Stepwise combinatorial assembling

[edit] Split-&-Pool DNA Encoding

In order to apply combinatorial chemistry for the synthesis of DNA-encoded chemical libraries, a Split-&-Pool approach was pursued. Initially a set of unique DNA-oligonucleotides (n) each containing a specific coding sequence is chemically conjugated to a corresponding set of small organic molecules.Consequently the oligonucleotide-conjugate compounds are mixed ("Pool") and divided("Split")into a number of groups (m). In appropriate conditions a second set of building blocks (m) are coupled to the first one and a further oligonucleotide which is coding for the second modification is enzymatically introduced before mixing again. This “split-&-pool” steps can be iterated a number of times (r) increasing at each round the library size in a combinatorial manner (i.e. (n x m)^r).

[edit] DNA-routing

In 2004, D.R. Halpin and P.B. Harbury presented a novel intriguing method for the construction of DNA-encoded libraries. For the first time the DNA-conjugated templates served for both encoding and programming the infrastructure of the “split-&-pool” synthesis of the library components.^[6] 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.^[6] According to this split-and-pool protocol a peptide combinatorial library DNA-encoded of 10⁶ members was generated.^[7]

[edit] DNA-templated synthesis

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 olgonucleotide 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.

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.^[8][9] A DNA-heteroduplex was used to accelerate the reaction between chemical moieties displayed at the extremities of the two DNA strands. Furthermore, the "proximity effect", which accelerates bimolecular reaction, was shown to be distance-independent (at least within a distance of 30 nucleotides).^[8][9]. In a sequence-programmed fashion oligonucleotides carrying one chemical reactant group were hybridized to complementary oligonucleotide derivatives carrying a different reactive chemical group. The close proximity conferred by the DNA hybridization drastically increases the effective molarity of the reaction reagents attached to the oligonucleotides, enabling the desired reaction to occur even in an aqueous environment at concentrations which are several orders of magnitude lower than those needed for the corresponding conventional organic reaction not DNA-templated.^[10]. Using a DNA-templated set-up and sequence-programmed synthesis Liu and co-workers generated a 64 member compound DNA encoded library of macrocycles.^[11]

[edit] Stepwise coupling of coding DNA fragments to nascent organic molecules

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.

A promising strategy for the construction of DNA-encoded libraries is represented by the use of multifunctional building blocks covalently conjugate to an oligonucleotide serving as a “core structure” for library synthesis. In a ‘pool-and-split’ fashion a set of multifunctional scaffolds undergo orthogonal reactions with series of suitable reactive partners. Following each reaction step, the identity of the modification is encoded by an enzymatic addition of DNA segment to the original DNA “core structure”.^[12][13] The use of N-protected amino acids covalently attached to a DNA fragment allow, after a suitable deprotection step, a further amide bond formation with a series of carboxylic acids or a reductive amination with aldehydes. 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. After completion of the desired reaction step, the identity of the chemical moiety added to the oligonucleotide is established by the annealing of a partially complementary oligonucleotide and by a subsequent Klenow fill-in DNA-polymerization, yielding a double stranded DNA fragment. 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.^[12](Fig.2)

[edit] Combinatorial self-assembling

[edit] Encoded Self-Assembling Chemical libraries

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).

Encoded Self-Assembling Chemical (ESAC) libraries rely on the principle that two sublibraries of a size of x members (e.g. 10³) containing a constant complementary hybridization domain can yield a combinatorial DNA-duplex library after hybridization with a complexity of x² uniformly represented library members (e.g. 10⁶).^[14] Each sub-library member would consist of an oligonucleotide containing a variable, coding region flanked by a constant DNA sequence, carrying a suitable chemical modification at the oligonucleotide extremity.^[14] The ESAC sublibraries can be used in at least four different embodiments.^[14]

• A sub-library can be paired with a complementary oligonucleotide and used as a DNA encoded library displaying a single covalently linked compound for affinity-based selection experiments.
• A sub-library can be paired with an oligonucleotide displaying a known binder to the target, thus enabling affinity maturation strategies.
• Two individual sublibraries can be assembled combinatorially and used for the de novo identification of bindentate binding molecules.
• Three different sublibraries can be assembled to form a combinatorial triplex library.

Preferential binders isolated from an affinity-based selection can be PCR-amplified and decoded on complementary oligonucleotide microarrays^[15] or by concatenation of the codes, subcloning and sequencing^[16]. The individual building blocks can eventually be conjugated using suitable linkers to yield a drug-like high-affinity compound. The characteristics of the linker (e.g. length, flexibility, geometry, chemical nature and solubility) influence the binding affinity and the chemical properties of the resulting binder.(Fig.3)

Bio-panning experiments on HSA of a 600-member ESAC library allowed the the isolation of the 4-(p-iodophenyl)butanoic moiety. The compound represents the core structure of a series of portable albumin binding molecules and of Albufluor^TM a recently developed fluorescein angiographic contrast agent currently under clinical evaluation.^[17]

ESAC technology has been used for the isolation of potent inhibitors of bovine trypsin and for the identification of novel inhibitors of stromelysin-1 (MMP-3) , a matrix metalloproteinase involved in both physiological and pathological tissue remodeling processes, as well as in disease processes, such as arthritis and metastasis.^[18][19]

[edit] Decoding of DNA-encoded chemical libraries

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. So far, Sanger-sequencing-based decoding, microarray-based methodology and high-throughput sequencing techniques represented the main methodologies for the decoding of DNA-encoded library selections.

[edit] Sanger sequencing-based decoding

Although many authors implicitly envisaged a traditional Sanger sequencing-based decoding,^{[3][4][7][11][14]} the number of codes to sequence simply according to the complexity of the library is definitely an unrealistic task for a traditional Sanger sequencing approach. Nevertheless, the implementation of Sanger sequencing for decoding DNA-encoded chemical libraries in high-throughput fashion was the first to be described.^[14] 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.^[14]

[edit] Microarray-based decoding

A DNA microarray is a device for high-throughput investigations widely used in molecular biology and in medicine. It consists of an arrayed series of microscopic spots (‘features’ or ‘locations’) containing few picomoles of oligonucleotides carrying a specific DNA sequence. This can be a short section of a gene or other DNA element that are used as probes to hybridize a DNA or RNA sample under suitable conditions. Probe-target hybridization is usually detected and quantified by fluorescence-based detection of fluorophore-labeled targets to determine relative abundance of the target nucleic acid sequences. Microarray has been used for the successfully decoding of ESAC DNA-encoded libraries.^[14] The coding oligonucleotides representing the individual chemical compounds in the library, are spotted and chemically linked onto the microarray slides, using a BioChip Arrayer robot. 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. Afterwards, microarrays are analyzed using a laser scan and spot intensities detected and quantified. The enrichment of the preferential binding compounds is revealed comparing the spots intensity of the DNA-microarray slide before and after selection.^[14]

[edit] Decoding by high throughput sequencing

According to the complexity of the DNA encoded chemical library (typically between 10³ and 10⁶ members), a conventional Sanger sequencing based decoding is unlikely to be usable in practice, due both to the high cost per base for the sequencing and to the tedious procedure involved.^[20] High throughput sequencing technologies exploited strategies that parallelize the sequencing process displacing the use of capillary electrophoresis and producing thousands or millions of sequences at once. In 2008 was described the first implementation of a high-throughput sequencing technique originally developed for genome sequencing (i.e. "454 technology") to the fast and efficient decoding of a DNA encoded chemical library comprising 4000 compounds.^[12] This study led to the identification of novel chemical compounds with submicromolar dissociation constants towards streptavidin and definitely shown the feasibility to construct, perform selections and decode DNA-encoded libraries containing millions of chemical compounds.^[12]

[edit] See also

• Drug discovery
• High-throughput screening
• Combinatorial chemistry
• DNA sequencing
• Phage display

[edit] References