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| title | chunk | source | category | tags | date_saved | instance |
|---|---|---|---|---|---|---|
| DNA-encoded chemical library | 5/6 | https://en.wikipedia.org/wiki/DNA-encoded_chemical_library | reference | science, encyclopedia | 2026-05-05T10:04:16.238098+00:00 | kb-cron |
== 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 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.
=== 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 and PNA-encoded libraries. 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.
=== 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. 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. 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.
== Alternative barcodes to DNA == DNA barcodes can have limitations. Many standard chemical reactions can degrade DNA and thus compromise the chemical barcodes, necessitating changing chemical reaction conditions that could alter the binding ability of the small molecule to its target. Additionally, the DNA tag is typically over 50 times larger than the molecule itself, potentially restricting the binding ability of each library member and sometimes interacting with the target itself, creating false hits or obscuring potentially otherwise strong binders . This is especially problematic when the target has nucleic acid binding sites, like transcription factors or RNA-binding proteins. For this reason, a multitude of barcode alternatives have been developed in efforts to mitigate these issues such as abiotic peptides , peptide nucleic acids, and even barcode free self-encoded libraries.
=== Abiotic peptides === In contrast to DNA-encoded chemical libraries (DELS), abiotic peptide-encoded libraries (PELs) are emerging as an alternative in which synthetic peptide sequences are used as carriers of chemical information and small molecule discovery. Whereas DELs rely on nucleic acid tags and PCR amplification, PELs use non-natural amino acid sequences to store information which can be decoded by tandem mass spectrometry (MS/MS) . In a protein-encoded library system, small molecules are synthesized using a split-and-pool strategy while being covalently linked to a peptide tag that is elongated orthogonally to encode each split step. This results in a peptide sequence that functions as a molecular barcode for information storage, similar to how DNA functions in DELs. The use of non-natural amino acids with distinct mass spectrometry signatures allows for the store of sequence-defined information that can be decoded after affinity selection against protein targets . The motivation behind the development of abiotic peptide encoding is the limited chemical reaction compatibility of DNA, which can degrade under various conditions . Since abiotic peptide encoding does not require DNA, these tags exhibit a greater library of molecules that can be synthesized and are also compatible with a broader range of chemical reactions . Despite this work, PELs still has its limitations, as PEL library sizes are orders of magnitude smaller than current DELs. Additionally, PELs lack the inherent amplification capabilities of DELs and rely on current mass spectrometric analysis for decoding. Since peptide decoding requires complex mass spectrometry and computational power, this means that current detection sensitivity limits practical PEL sizes .
=== Peptide nucleic acid (PNA) encoded libraries ===