Global InformationSystematic evolution of ligands by exponential enrichment information
Systematic evolution of ligands by exponential enrichment (SELEX), also referred to as in vitro selection or in vitro evolution, is a combinatorial chemistry technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. These single-stranded DNA or RNA are commonly referred to as aptamers.[1][2][3] Although SELEX has emerged as the most commonly used name for the procedure, some researchers have referred to it as SAAB (selected and amplified binding site) and CASTing (cyclic amplification and selection of targets)[4][5] SELEX was first introduced in 1990. In 2015, a special issue was published in the Journal of Molecular Evolution in the honor of quarter century of the discovery of SELEX.[6]
The process begins with the synthesis of a very large oligonucleotide library, consisting of randomly generated sequences of fixed length flanked by constant 5' and 3' ends. The constant ends serve as primers, while a small number of random regions are expected to bind specifically to the chosen target. For a randomly generated region of length n, the number of possible sequences in the library using conventional DNA or RNA is 4n (n positions with four possibilities (A,T,C,G) at each position). The sequences in the library are exposed to the target ligand - which may be a protein or a small organic compound - and those that do not bind the target are removed, usually by affinity chromatography or target capture on paramagnetic beads.[7] The bound sequences are eluted and amplified by PCR[2][3] to prepare for subsequent rounds of selection in which the stringency of the elution conditions can be increased to identify the tightest-binding sequences.[2] A caution to consider in this method is that the selection of extremely high, sub-nanomolar binding affinity entities may not in fact improve specificity for the target molecule.[8] Off-target binding to related molecules could have significant clinical effects.
SELEX has been used to develop a number of aptamers that bind targets interesting for both clinical and research purposes.[9] Nucleotides with chemically modified sugars and bases have been incorporated into SELEX reactions to increase the chemical diversity at each base, expanding the possibilities for specific and sensitive binding, or increasing stability in serum or in vivo.[9][10]
- ^ Hak-Hagir A (1978). "[The Hak-Hagir skin conduit]". Zeitschrift für Urologie und Nephrologie. 71 (9): 639–642. PMID 362762.
- ^ a b c Tuerk C, Gold L (August 1990). "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase". Science. 249 (4968): 505–10. Bibcode:1990Sci...249..505T. doi:10.1126/science.2200121. PMID 2200121.
- ^ a b Ellington AD, Szostak JW (August 1990). "In vitro selection of RNA molecules that bind specific ligands". Nature. 346 (6287): 818–22. Bibcode:1990Natur.346..818E. doi:10.1038/346818a0. PMID 1697402. S2CID 4273647.
- ^ Blackwell TK, Weintraub H (November 1990). "Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection". Science. 250 (4984): 1104–10. Bibcode:1990Sci...250.1104B. doi:10.1126/science.2174572. PMID 2174572. S2CID 1995608.
- ^ Wright WE, Binder M, Funk W (August 1991). "Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site". Molecular and Cellular Biology. 11 (8): 4104–10. doi:10.1128/mcb.11.8.4104. PMC 361222. PMID 1649388.
- ^ Gold L (December 2015). "SELEX: How It Happened and Where It will Go". Journal of Molecular Evolution. 81 (5–6): 140–143. Bibcode:2015JMolE..81..140G. doi:10.1007/s00239-015-9705-9. PMC 4661202. PMID 26480964.
- ^ Stoltenburg R, Schubert T, Strehlitz B (2015-07-29). "In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A". PLOS ONE. 10 (7): e0134403. Bibcode:2015PLoSO..1034403S. doi:10.1371/journal.pone.0134403. PMC 4519192. PMID 26221730.
- ^ Carothers JM, Oestreich SC, Szostak JW (June 2006). "Aptamers selected for higher-affinity binding are not more specific for the target ligand". Journal of the American Chemical Society. 128 (24): 7929–37. doi:10.1021/ja060952q. PMC 4287982. PMID 16771507.
- ^ a b Wu YX, Kwon YJ (August 2016). "Aptamers: The "evolution" of SELEX". Methods. 106: 21–8. doi:10.1016/j.ymeth.2016.04.020. PMID 27109056.
- ^ Keefe AD, Cload ST (August 2008). "SELEX with modified nucleotides". Current Opinion in Chemical Biology. 12 (4): 448–56. doi:10.1016/j.cbpa.2008.06.028. PMID 18644461.