Directed evolution of proteins and nucleic acids using nonhomologous random recombination
The nonhomologous random recombination (NRR) method allows portions of nucleic acids to be recombined at sites where there is little or no sequence homology. This increases the frequency at which novel sequences are generated, allowing a more efficient and complete exploration of nucleic acid and protein diversity. NRR is a simple method that diversifies proteins in ways that are difficult or impossible to achieve by existing methods. Studies have shown that NRR could drive the production of hybrid enzymes with activity greater than those hybrids formed by traditional DNA shuffling. The implementation of protein NRR is straightforward, enabling starting DNA to be converted into a diversified library in about 1 day. In addition to applications for evolving proteins, NRR is useful for evolving nucleic acids with catalytic or binding (aptamer) activities.
Comparison of diversification methods for nucleic acid evolution Starting with parental sequences, pure SELEX enriches active sequences. Error-prone PCR yields parental sequences with point mutations. Homologous recombination methods, such as DNA shuffling, allow crossovers between sequences only in regions of homology. Nonhomologous random recombination allows many other changes, including random crossovers between any sequences, repetition, reordering, reorienting, elimination of subsequences, or any combination of these changes.
The NRR method for proteins One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry to the ligation reaction to generate recombined products of the desired average size. The ends of the hairpins are removed by restriction digestion, and the PCR-amplified pool is cloned for protein expression and selection.
Intellectual Property Status: Patent(s) Pending
Protein engineering by laboratory directed molecular evolution has proven useful in conferring desired properties upon a wide variety of commercially important biomolecules. Successful evolution requires the efficient exploration of sequence space to locate molecules encoding the desired properties. Previous methods of introducing diversity include whole-genome mutagenesis, error-prone PCR, random cassette mutagenesis, and DNA shuffling. Of these, only DNA shuffling allows the recombination of parent DNA sequences, but it is still limited to recombination at sites of homology between sequences.