An in vitro translation, selection, and amplification system for peptide nucleic acids

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) that integrates a carefully designed "genetic code" of twelve PNA pentanucleotide building block, an optimized DNA-templated PNA polymerization, a strategy for display of single-stranded PNA polymers, and molecular biology methods to prepare DNA templates surviving in vitro selection and PCR amplification for subsequent rounds of translation and selection. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 x 10 8 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (avidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory.