Multi-state surface engineering improves Phi29 solubility, thermostability, and low-input amplification

Phi29 DNA polymerase is widely used for isothermal whole-genome amplification but is limited by low-salt aggregation, modest thermostability, and inconsistent performance with low-input DNA. Here, we developed a multi-state surface-charge engineering workflow that combines Pythia-based stability scoring with AlphaFold3 structural filtering across apo and DNA-bound states. Screening solvent-exposed aromatic residues yielded two matched eight-site variants, Phi29-M1 and Phi29-M2. Both variants retained rolling circle amplification activity while showing approximately 3-fold higher soluble recovery under low-salt stress, 2°C–3°C higher melting temperatures, and 2- to 3-fold stronger DNA binding, mainly through faster association. In multiple-displacement amplification, M1 and M2 generated usable low-template input (10 pg) products at 30°C–37°C under conditions where wild-type Phi29 failed. Molecular dynamics simulations were consistent with reduced flexibility around the exonuclease-palm and fingers-side tunnel regions. Together, these findings support the potential utility of multi-site surface engineering for improving Phi29 robustness in low-input genomic applications.