DNA-ENCODED CHEMICAL LIBRARIES (DEL) is a technology for the synthesis and screening on unprecedented scale of collections of small molecule compounds. DEL is used in medicinal chemistry to bridge the fields of combinatorial chemistry and molecular biology . The aim of DEL technology is to accelerate the drug discovery process and in particular early phase discovery activities such as target validation and hit identification.
DEL technology involves the conjugation of chemical compounds or
building blocks to short
* 1 DNA-encoded chemical libraries and display technologies * 2 History
* 3 Non-evolution based technologies
* 3.1 Split-"> 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. 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. 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- through rounds of selection, amplification and translation.
NON-EVOLUTION BASED TECHNOLOGIES
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-"> 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-"> 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). Each
sub-library member would consist of an oligonucleotide containing a
variable, coding region flanked by a constant
* A sub-library can be paired with a complementary oligonucleotide
and used as a
Preferential binders isolated from an affinity-based selection can be PCR-amplified and decoded on complementary oligonucleotide microarrays or by concatenation of the codes, subcloning and sequencing . 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 isolation of the 4-(p-iodophenyl)butanoic moiety. The compound represents the core structure of a series of portable albumin binding molecules and of AlbufluorTM a recently developed fluorescein angiographic contrast agent currently under clinical evaluation.
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 .
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-"> 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.
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 .
A DNA-heteroduplex was used to accelerate the reaction between
chemical moieties displayed at the extremities of the two
3-DIMENSIONAL PROXIMITY-BASED TECHNOLOGY (YOCTOREACTOR TECHNOLOGY)
THE YOCTOREACTOR (YR) is a 3D proximity-driven approach which
exploits the self-assembling nature of
The center of the
BUILDING A YR LIBRARY
Figure 6 illustrates the generation of a yR library using a 3-way DNA
junction. 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
In summary, chemical building-blocks (BB) are attached via cleavable
or non-cleavable linkers to three types of bispecific DNA
oligonucleotides (oligo-BBs) representing each arm of the yR. To
facilitate synthesis in a combinatorial manner, the oligo-BBs are
designed such that the
The yR design approach provides an unvarying reaction site with regard to both (a) distance between reactants and (b) sequence environment surrounding the reaction site. 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. The code of the synthesized products, furthermore, is not preset, but rather is assembled combinatorially and synthesized in synchronicity with the innate product.
HOMOGENEOUS SCREENING OF YOCTOREACTOR LIBRARIES
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. Called Binder Trap
Enrichment (BTE), ligands to a protein target are identified by
trapping binding pairs (DNA-labelled protein target and yR ligand) in
emulsion droplets during dissociation dominated kinetics. Once
trapped, the target and ligand
Hereafter, identification of hits is essentially a counting exercise:
information on binding events is deciphered by sequencing and counting
BTE mimics the non-equilibrium nature of in vivo ligand-target interactions and offers the unique possibility to screen for target specific ligands based on ligand-target residence time because the emulsion, which traps the binding complex, is formed during a dynamic dissociation phase.
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.
SANGER SEQUENCING-BASED DECODING
Although many authors implicitly envisaged a traditional Sanger
sequencing -based decoding, 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. After selection and
DECODING BY HIGH THROUGHPUT SEQUENCING
According to the complexity of the
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