Elucidating the Determinants of Polymerase Specificity by Microfluidic Based Deep Mutational Scanning.
Engineering polymerases to synthesize artificial genetic polymers with unique backbone structures is limited by a general lack of understanding about the structural determinants that govern substrate specificity. Here, we report a high throughput microfluidic based approach for mapping sequence function relationships that combines droplet based optical polymerase sorting with deep mutational scanning. We applied this strategy to map the finger subdomain of a replicative DNA polymerase isolated from Thermococcus kodakarensis (Kod) The enrichment profile provides an unbiased view of the ability of each mutant to synthesize threose nucleic acid, which was used as a model non natural genetic polymer. From a single round of sorting, we discovered two cases of positive epistasis and demonstrate the near inversion of substrate specificity from a double mutant variant. This effort indicates that polymerase specificity may be governed by a small number of highly specific residues that can be elucidated by deep mutational scanning without the need for iterative rounds of directed evolution.
ACS synthetic biology