Directed evolution: overcoming biology's limitations

Abstract

A process in which peptide nucleic acids may be used for in vitro evolution has been developed. This method can offer enormous opportunities to evolve stable, non-natural molecules for therapeutic applications.

Figure 1

Scheme for in vitro evolution using peptide nucleic acids (PNAs). For the translation step (a), PNA pentamers with reactive aldehydes and amines on the termini are condensed in a sequence-dependent manner on a DNA template using reductive amination. This step uses a mixture of DNA templates to generate a library of PNAs bound to their complementary DNA sequences. For displacement (b), Herculase II polymerase is used to make DNA duplexes and dislocate the PNAs from the DNA templates. Selection (c) is then performed followed by isolation and amplification (d). The process can be iterated several times to achieve enrichment for streptavidin binding of a biotin-labeled PNA present in the library at a 1:10⁶ ratio. While the current process is a proof-of-concept, this scheme lays the foundation to explore PNA-based evolution for biologically important protein targets.