Directed evolution of polymerase function by compartmentalized self-replication

We describe compartmentalized self-replication (CSR), a strategy for the di rected evolution of enzymes, especially polymerases. CSR is based an a simp le feedback loop consisting of a polymerase that replicates only its own en coding gene. Compartmentalization serves to isolate individual self-replica tion reactions from each other. In such a system, adaptive gains directly ( and proportionally) translate into genetic amplification of the encoding ge ne. CSR has applications in the evolution of polymerases with novel and use ful properties. By using three cycles of CSR, we obtained variants of Tag D NA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reacti on allows its application to enzymes other than polymerases. We show that n ucleoside diphosphate kinase and Tag polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate ki nase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptid es cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases , as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.