Engineering polymerases for applications in synthetic biology.

DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

Evaluating the Rate and Substrate Specificity of Laboratory Evolved XNA Polymerases.

Engineered polymerases that can copy genetic information between DNA and xeno-nucleic acids (XNA) hold tremendous value as reagents in future biotechnology applications. However, current XNA polymerases function with inferior activity relative to their natural counterparts, indicating that current polymerase engineering efforts would benefit from new benchmarking assays. Here, we describe a highly parallel, low-cost method for measuring the average rate and substrate specificity of XNA polymerases in a standard qPCR instrument. Our approach, termed polymerase kinetic profiling (PKPro), involves monitoring XNA synthesis on a self-priming template using high-resolution melting (HRM) fluorescent dyes that intercalate into the growing duplex as the template strand is copied into XNA. Since changes in fluorescence are directly proportional to XNA synthesis, quantitative measurements are obtained by calibrating the fluorescent signal against chemically synthesized standards. Using PKPro, we discovered that XNA polymerases function with rates of ∼1-80 nt/min and exhibit substrate specificities of ∼0.1-5-fold for xNTP versus dNTP. Last, we show how PKPro could be used in a highly parallel screen by analyzing 288 different polymerase reaction conditions. On the basis of these results, we suggest that PKPro provides a powerful tool for evaluating the activity of XNA polymerases.

Directed evolution of custom polymerases using droplet microfluidics.

DNA polymerases are critical tools for a large number of emerging applications in biotechnology, but oftentimes polymerases with desired functions are not readily available. Directed evolution provides a possible solution to this problem by enabling the creation of engineered polymerases that are better equipped to recognize a given unnatural substrate. Here we report a microfluidic-based method for evolving new polymerase functions that involves ultrahigh throughput sorting of fluorescent water-in-oil (w/o) microdroplets. The workflow entails the expression of a diverse population of polymerase variants in E. coli, production of microfluidic droplets containing one or less E. coli, bacteria lysis to release the polymerase and encoding plasmid into the surrounding droplet, a fluorescence-based activity assay to identify variants with a desired activity, isolation of fluorescent droplets using a fluorescence activated droplet sorting (FADS) device, and plasmid recovery with DNA sequencing to determine the identity of the functional variants. This technique is amenable to any type of unnatural nucleic acid and/or polymerase function, including DNA-templated synthesis, reverse transcription, and replication.

Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution.

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

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.

Fluorescence-Activated Droplet Sorting for Single-Cell Directed Evolution.

Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescence-activated droplet sorting (FADS) instrument that was established to evolve enzymes for synthesizing and modifying artificial genetic polymers (XNAs). The microfluidic system enables droplet sorting at ∼2-3 kHz using fluorescent sensors that are responsive to enzymatic activity. The ability to evolve nucleic acid enzymes with customized properties will uniquely drive emerging applications in synthetic biology, biotechnology, and healthcare.

Evolution of a General RNA-Cleaving FANA Enzyme.

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics. Here, we describe an efficient RNA-cleaving 2'-fluoroarabino nucleic acid enzyme (FANAzyme) that functions with a rate enhancement of >106-fold over the uncatalyzed reaction and exhibits substrate saturation kinetics typical of most natural enzymes. The FANAzyme was generated by in vitro evolution using natural polymerases that were found to recognize FANA substrates with high fidelity. The enzyme comprises a small 25 nucleotide catalytic domain flanked by substrate-binding arms that can be engineered to recognize diverse RNA targets. Substrate cleavage occurs at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Our results expand the chemical space of nucleic acid enzymes to include nuclease-resistant scaffolds with strong catalytic activity.

Engineered Polymerases with Altered Substrate Specificity: Expression and Purification.

Polymerase engineering is making it possible to synthesize xeno-nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which aims to study the genetic concepts of heredity and evolution in artificial genetic polymers. Since many of the polymerases needed to synthesize XNA polymers are not available commercially, researchers must express and purify these enzymes as recombinant proteins from E. coli. This unit details the steps needed to express, purify, and evaluate the activity of engineered polymerases with altered substrate recognition properties. The protocol requires 6 days to complete and will produce ∼20 mg of pure, nuclease-free polymerase per liter of E. coli bacterial culture. © 2017 by John Wiley & Sons, Inc.

Structural basis for TNA synthesis by an engineered TNA polymerase.

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

DNA sequencing using polymerase substrate-binding kinetics.

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.