Systematic Evolution of Ligands by Exponential Enrichment

SELEX ("Systematic Evolution of Ligands by Exponential Enrichment"), also referred to as "in vitro selection" or "in vitro evolution", is a combinatorial technique in molecular biology for producing oligonucleotides of either single-stranded DNA or RNA that specifically bind to a target ligand or ligands. [Ellington, A. D. & Szostak, J. W. (1990) Nature (London) 346, 818-822] [Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. ] The selected sequences are referred to as aptamers.

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 that serve as primers. For a randomly generated region of length "n", the number of possible sequences in the library is 4ⁿ. The sequences in the library are exposed to the target ligand - which may be a protein or small organic compound - and those that do not bind the target are removed, usually by affinity chromatography. The bound sequences are eluted and amplified by RT-PCR to prepare for subsequent rounds of selection in which the stringency of the elution conditions is increased to identify the tightest-binding sequences. An advancement on the original method allows an RNA library to omit the constant primer regions, which can be difficult to remove after the selection process because they stabilize secondary structures that are unstable when formed by the random region alone.Jarosch F, Buchner K, Klussmann S. (2006). In vitro selection using a dual RNA library that allows primerless selection. "Nucleic Acids Res" 34(12):e86.]

The technique has been used to evolve aptamers of extremely high binding affinity to a variety of target ligands, including small molecules such as ATPDieckmann T, Suzuki E, Nakamura GK, Feigon J. (1996). Solution structure of an ATP-binding RNA aptamer reveals a novel fold. "RNA" 2(7):628-40.] and adenosineHuizenga DE, Szostak JW. (1995). A DNA aptamer that binds adenosine and ATP. "Biochemistry" 34(2):656-65.] Burke DH, Gold L. (1997). RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX. "Nucleic Acids Res" 25(10):2020-4.] and proteins such as prionsMercey R, Lantier I, Maurel MC, Grosclaude J, Lantier F, Marc D. (2006). Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns. "Arch Virol" 151(11):2197-214.] and vascular endothelial growth factor (VEGF).Ulrich H, Trujillo CA, Nery AA, Alves JM, Majumder P, Resende RR, Martins AH. (2006). DNA and RNA aptamers: from tools for basic research towards therapeutic applications. "Comb Chem High Throughput Screen" 9(8):619-32.] Clinical uses of the technique are suggested by aptamers that bind tumor markersFerreira CS, Matthews CS, Missailidis S. (2006). DNA aptamers that bind to MUC1 tumour marker: design and characterization of MUC1-binding single-stranded DNA aptamers. "Tumour Biol" 27(6):289-301.] and clinical trials are underway for a VEGF-binding aptamer trade-named Macugen in treating macular degeneration.Vavvas D, D'Amico DJ. (2006). Pegaptanib (Macugen): treating neovascular age-related macular degeneration and current role in clinical practice. "Ophthalmol Clin North Am." 19(3):353-60.]

One caution advanced in relation to the method emphasizes that selection for extremely high, sub-nanomolar binding affinities may not in fact improve specificity for the target molecule.Carothers JM, Oestreich SC, Szostak JW. (2006). Aptamers selected for higher-affinity binding are not more specific for the target ligand. "J Am Chem Soc" 128(24):7929-37.] Off-target binding to related molecules could have significant clinical effects.

References

Further reading

*cite journal |author=Levine HA, Nilsen-Hamilton M |title=A mathematical analysis of SELEX |journal=Computational biology and chemistry |volume=31 |issue=1 |pages=11–35 |year=2007 |pmid=17218151 |doi=10.1016/j.compbiolchem.2006.10.002