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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| DNA-encoded chemical library | 4/6 | https://en.wikipedia.org/wiki/DNA-encoded_chemical_library | reference | science, encyclopedia | 2026-05-05T10:04:16.238098+00:00 | kb-cron |
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 DNA strands. Furthermore, the "proximity effect", which accelerates bimolecular reaction, was shown to be distance-independent (at least within a distance of 30 nucleotides). In a sequence-programmed fashion oligonucleotides carrying one chemical reactant group were hybridized to complementary oligonucleotide derivatives carrying a different reactive chemical group. The 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. Using a DNA-templated set-up and sequence-programmed synthesis Liu and co-workers generated a 64-member compound DNA encoded library of macrocycles.
== 3-Dimensional proximity-based technology (YoctoReactor technology) == The YoctoReactor (yR) is a 3D proximity-driven approach which exploits the self-assembling nature of DNA oligonucleotides into 3, 4 or 5-way junctions to direct small molecule synthesis at the center of the junction. Figure 5 illustrates the basic concept with a 4-way DNA junction.
The center of the DNA junction constitutes a volume on the order of a yoctoliter, hence the name YoctoReactor. This volume contains a single molecule reaction yielding reaction concentrations in the high mM range. The effective concentration facilitated by the DNA greatly accelerates chemical reactions that otherwise would not take place at the actual concentration several orders of magnitude lower.
=== Building a yR library === Figure 6 illustrates the generation of a yR library using a 3-way DNA junction. 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 DNA contains (a) the code for an attached BB at the distal end of the oligo (colored lines) and (b) areas of constant DNA sequence (black lines) to bring about the self-assembly of the DNA into a 3-way junction (independently of the BB) and the subsequent chemical reaction. 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. Table 1 outlines how libraries of different sizes can be generated using yR technology.
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 DNA are joined by ligation, thus preserving the binding information. Hereafter, identification of hits is essentially a counting exercise: information on binding events is deciphered by sequencing and counting the joined DNA - selective binders are counted with a much higher frequency than random binders. This is possible because random trapping of target and ligand is "diluted" by the high number of water droplets in the emulsion. The low noise and background signal characteristic of BTE is attributed to the "dilution" of the random signal, the lack of surface artifacts and the high fidelity of the yR library and screening method. Screening is performed in a single tube method. Biologically active hits are identified in a single round of BTE characterized by a low false positive rate. 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.
=== Screening of DELs in cells === DNA-encoded libraries (DELs) have been adapted for screening in living cells to better reflect native biological conditions, specifically using Xenopus laevis oocytes. This approach, termed cellular Binder Trap Enrichment (cBTE), facilitates the identification of small-molecule ligands that bind to target proteins in a native cellular environment. In this method, the protein of interest (POI) is expressed in oocytes as a fusion with a "Prey" protein, such as carbonic anhydrase IX (CAIX). Simultaneously, a "Bait" molecule—comprising a known ligand for the Prey protein linked to a DNA strand—is introduced. Alongside the Bait, a DEL is co-injected into the oocytes. If a DEL member binds to the POI, it brings its attached DNA tag into the same molecular complex as the Bait DNA via the POI–Prey–Bait interaction. Following incubation, the oocytes are lysed, and the lysate is subjected to Binder Trap Enrichment (BTE) as described above. In essence, the DEL and Bait DNA are ligated in droplets, thus encoding the binding event. The ligated DNA is then amplified and subjected to high-throughput sequencing to identify the DEL members that interacted with the POI.
This intracellular screening technique allows for the discovery of ligands that engage targets in their native conformation and cellular environment, enabling screening of targets that are difficult to express or purify, and potentially improving the physiological relevance of identified compounds.