Directed evolution of Phi29 DNA Polymerase for HNA synthesis

Using the DNA polymerase from B. subtilis Phi29 bacteriophage (phi29 DNAP) as a model, we wanted to demonstrate how coupling focused libraries, selection and deep sequencing can be combined to identify variants of interest for characterization. We targeted three loops (TRP2: L406-L412, thumb: V559-V566, and exonuclease T15-V24) of a previously reported thermostabilized Phi29 DNA polymerase mutant harboring the additional D12A mutation (to disable exonuclease activity) through insertion and deletion (indel) mutagenesis. We subjected these libraries to selection via compartmentalized self-tagging (CST) for HNA (1,5-anhydrohexitol nucleic acid) synthesis. As selection parameters can be controlled, different areas of an enzyme mechanism can be explored through directed evolution selection strategies. We focused selection on faster HNA synthesis. We hypothesized that the interaction between these three loops is likely functional, capable of providing the dynamic response needed for polymerase-exonuclease balance and enzyme processivity. Additionally, given that the HNA-DNA heteroduplex is expected to be wider than a DNA duplex, we hypothesized that an insertion or deletion could lead to stabilizing interactions between the three subdomains, disrupted by the wider substrate, to being re-established.