We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA 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 reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase 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 polypeptides 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. P olynucleotide polymerases occupy a central role in the maintenance, transmission, and expression of genetic information (1). They also have enabled core technologies of molecular biology like sequencing, PCR, site-directed mutagenesis, and cDNA cloning. However, polymerases available from nature are often not optimally suited for these applications and attempts have been made to tailor polymerase function by using either design or selection strategies. Structural studies have greatly advanced our understanding of polymerase function (2-4) and together with insights gained from site-directed mutagenesis have allowed the rational design of some polymerase variants with improved properties. Among these are polymerases with improved dideoxynucleotide incorporation for cycle-sequencing (5) or increased processivity (6). Other ''designer'' polymerases include truncation variants (7,8), some of which show improved thermostability and fidelity, although at the cost of reduced processivity. Despite these advances, our ability to engineer designer polymerases for specific applications remains limited.Repertoire selection methods have proven to be an effective means to obtain biopolymers with desired properties. Polymerases have been selected successfully for activity by phage display (9) and by complementation of a DNA polymerase I-deficient Escherichia coli strain (10). Careful screening of the complementing polymerase mutants yielded polymerases with a range of interesting properties such as alt...

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DNA polymerases recognize their substrates with exceptionally high specificity, restricting the use of unnatural nucleotides and the applications they enable. We describe a strategy to expand the substrate range of polymerases. By selecting for the extension of distorting 3' mismatches, we obtained mutants of Taq DNA polymerase that not only promiscuously extended mismatches, but had acquired a generic ability to process a diverse range of noncanonical substrates while maintaining high catalytic turnover, processivity and fidelity. Unlike the wild-type enzyme, they bypassed blocking lesions such as an abasic site, a thymidine dimer or the base analog 5-nitroindol and performed PCR amplification with complete substitution of all four nucleotide triphosphates with phosphorothioates or the substitution of one with the equivalent fluorescent dye-labeled nucleotide triphosphate. Such 'unfussy' polymerases have immediate utility, as we demonstrate by the generation of microarray probes with up to 20-fold brighter fluorescence.

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