In vitro evolution of novel polymerases
Although KF and Taq DNA polymerases are able to incorporate unnatural bases into duplex DNA, the overall selectivity and kinetics for unnatural base replication are not yet sufficient for our long term goal of an expanded genetic code. For this purpose, the DNA polymerases need to be engineered to better recognize unnatural bases.
We have developed an activity-based selection system to evolve polymerases with novel activities (see diagram, below). The selection system involves the co-display on phage of DNA polymerase libraries and an oligonucleotide substrate that primes synthesis with an attached template. If a displayed polymerase mutant recognizes the attached substrate and the provided dNTPs, then it will extend the primer, and only then covalently attach a biotin-dUTP. Biotinylated phage particles may be selectively recovered using a streptavidin solid support. Since the provided substrates may include nucleotides that form unnatural base pairs, the selections may be run so that only those mutants that both efficiently synthesize the unnatural base pair, and then continue synthesis are biotinylated and recovered. We are now interested in using the system to evolve polymerases that will efficiently replicate DNA containing our unnatural base pairs, as well as polymerases that have properties optimized for other biotechnology applications such as genome sequencing or in vitro selections.
Examples of our evolved polymerases:
- Evolving a DNA polymerase into an RNA polymerase
- Evolving a DNA polymerase with bona fide unnatural activity: the ability to incorporate 3'-OMe ribonucleosides
- Evolving DNA polymerases for next generation DNA sequencing
- Initial efforts toward evolving a DNA polymerase that more efficiently recognizes DNA containing unnatural base pairs: the PICS self-pair
- Evolving a polymerase for next generation sequencing