A general overview of in vitro selection protocol. NA stands for Nucleic Acids (DNA, RNA, PNA) which start as a random pool, and are enriched through the selection process.

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.^[1][2] 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.^[3]

The technique has been used to evolve aptamers of extremely high binding affinity to a variety of target ligands, including small molecules such as ATP^[4] and adenosine^[5][6] and proteins such as prions^[7] and vascular endothelial growth factor (VEGF).^[8] Clinical uses of the technique are suggested by aptamers that bind tumor markers^[9] and clinical trials are underway for a VEGF-binding aptamer trade-named Macugen in treating macular degeneration.^[8][10]

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.^[11] Off-target binding to related molecules could have significant clinical effects.

References

Further reading

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