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SELEX or Systematic Evolution of Ligands by Exponential Enrichment is the oldest technique among all-in vitro selection methods. It has been developed to find tighter nucleic acid binders to any protein and better understand the principles of this interaction. It dates back to 1990 with reports from Ellington&Szostak ( and Tuerk&Gold ( Since then it has been developed into a mature method with a wide range of its varieties which include in the most advanced in vivo-SELEX (, and cell-SELEX ( Similar to other in vitro approaches it allows screening of libraries up to 1015 members. It is mainly limited to the selection of nucleic acids aptamers - natural (RNA or DNA) or synthetic (Xenonucleic acids or XNAs).


A typical library would contain 1015 individual oligonucleotides each of which is composed of a random region (20–50 nt) flanked by two standard primer binding sites for subsequent reverse transcription (if needed) and PCR amplification. In the selection step, this pool is incubated with target molecules, which may be attached to the surface (chromatography sorbent or nitrocellulose filter). After incubation, the column is washed and those oligonucleotides that are retained get eluted. A target-bound pool is then amplified in (RT-)PCR and this new enriched library is used in the next round of selection. Ten or more selection rounds are performed and the final library is NGS-screened for enriched motifs. Ultimately, the most representative members in the final library are further characterised in terms of their binding to a bait molecule.

Challenges and requirements

There are main challenges to this method:

The bait molecule is typically attached on the resin which introduces a steric hindrance and biases the selection Nuclease degradation is an issue in RNA and DNA especially for SELEX performed in vivo. Typically selection is performed out of the context in vitro, whereas obtained aptamers are often aimed at use in vivo. This issue has been lately addressed in multiple reports (

Future directions

Overall, SELEX is an umbrella term for a wide variety of methods. It is a good technique to introduce oneself into the in vitro selection world. In its simplest form, it requires only a minimal set of instruments and materials. More advanced versions of SELEX (e.g. in vivo) are obviously harder to implement, but developments in this field are highly important for the diagnostic and therapeutic use of aptamers and thus streamlining of these protocols is expected.