Investigating the Determinants of Polymerase Specificity by Droplet Microfluidics

University Of California-Irvine, Irvine CA

Investigators

Abstract

Every living organism on our planet requires a DNA polymerase to faithfully and efficiently copy its genetic information (DNA) during cell division. The ability to harness DNA synthesis in applications such as the polymerase chain reaction has revolutionized medical diagnostics, forensics and molecular biology. Future technological advances will require the customization of DNA polymerases for particular properties and particular applications. The goal of this project is to determine how DNA polymerases recognize their DNA targets and to use this information to customize a polymerase for recognition and copying of novel targets. This project also contains a significant educational component that is designed to attract and maintain student interest in the biological sciences. Engineering DNA polymerases to synthesize any type of unnatural nucleic acid polymer with the same catalytic efficiency as their natural counterparts will require a better understanding of how a polymerase sequence relates to its biochemical function. In this proposal, we will address this problem by applying a convergent science approach that combines high-throughput microfluidic screening with next-generation sequencing to assess the functional activity of all possible single-point mutations found in the catalytic domain of a replicative DNA polymerase. The large-scale sequencing data that results will be organized into protein fitness maps that reveal the functional consequences of each mutation (beneficial, neutral, or negative) against a defined set of synthetic analogs. Mutations that promote strong synthesis activity will be recombined and taken through stringent screens to create new polymerases with superior catalytic activity. The best enzymes will be characterized using kinetic, biochemical, and structural methods that define the rate, fidelity, and mechanism of synthesis. Successful completion of this study will provide new fundamental knowledge about the limits of polymerase engineering using cutting-edge microfluidic techniques that allow for massively parallel screening of novel polymerase activities inside water-in-oil microdroplets. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.