Evolving Aptamers with Unnatural Base Pairs

A novel technology, genetic alphabet expansion, has rapidly advanced through the successful creation of unnatural base pairs that function as a third base pair in replication. Recently, genetic alphabet expansion has been applied to some practical areas. Among them, the application to DNA aptamer generation is a good example of the broad utility of this technology. A hydrophobic unnatural base pair, Ds–Px, which exhibits high fidelity in replication as a third base pair, has been applied to an evolutionary engineering method called SELEX (Systematic Evolution of Ligands by EXponential enrichment) to generate DNA aptamers that bind to targets. A few Ds bases in DNA aptamers significantly increase the binding affinity to targets, enabling the use of DNA aptamers as an alternative to antibodies. This protocol describes the ExSELEX (genetic alphabet Expansion for SELEX) method to generate Ds-containing DNA aptamers. © 2017 by John Wiley & Sons, Inc.