Compartmentalized Self-replication Research Articles

Emulsion-based directed evolution of enzymes and proteins in yeast.

Emulsion-based selections are a unique type of directed evolution method that overcome common bottlenecks associated with purely in vivo selections. For example, emulsions including cell-free translation machinery can be useful for expression of toxic genes. However, not all cell types can efficiently produce protein in vitro, for example, the eukaryotic microbe Saccharomyces cerevisiae. compartmentalized self replication (CSR) and compartmentalized partnered replication (CPR) are two emulsion-based selection schemes that leverage the advantages of both in vivo and in vitro selections by compartmentalizing cells in water-in-oil droplets. Previous implementations of these methods utilized bacterial hosts, which has limited the technology to the directed evolution of proteins that can be heterologously expressed in prokaryotic systems. To expand the repertoire of targets that can be evolved, we have adapted emulsion-based PCR selections to be compatible with a eukaryotic host.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5′ to 3′ exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol.

Compartmentalized Self-Replication for Evolution of a DNA Polymerase.

Compartmentalized self-replication (CSR) is an emulsion PCR-based method for the selection of DNA polymerases. E. coli host cells expressing a library of DNA polymerases are emulsified so that no more than a single cell is present in a single emulsion droplet. In a subsequent emulsion PCR step, the DNA polymerase protein, as well as the plasmid encoding it are released into the emulsion droplet and the genes that created the most active or abundant polymerase variants are exponentially amplified and can be passed to the next round of CSR. CSR is a powerful method for engineering of polymerases since it allows selection under a variety of conditions, including the use of non-standard substrates. In this unit, we provide a step-by-step procedure for the selection of polymerases, using as an example the selection of reverse transcriptase activity starting from a library of Thermococcus kodakaraensis (KOD) DNA polymerase variants. © 2018 by John Wiley & Sons, Inc.

In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique.

Compartmentalized self replication (CSR) is widely used for in vitro evolution of thermostable DNA polymerases able to perform PCR in emulsion. We have modified and adapted CSR technique for isothermal DNA amplification using mezophilic phi29 DNA polymerase and whole genome amplification (WGA) reaction. In standard CSR emulsified bacterial cells are disrupted during denaturation step (94-96°C) in the first circles of PCR. Released plasmid DNA that encodes target polymerase and the thermophilic enzyme complement the emulsified PCR reaction mixture and start polymerase gene amplification. To be able to select for mezophilic enzymes we have employed multiple freezing-thawing cycles of emulsion as a bacterial cell wall disruption step instead of high temperature incubation. Subsequently WGA like plasmid DNA amplification could be performed by phi29 DNA polymerase applying different selection pressure conditions (temperature, buffer composition, modified dNTP, time, etc.). In our case the library of random phi29 DNA polymerase mutants was subjected to seven selection rounds of isothermal CSR (iCSR). After the selection polymerase variant containing the most frequent mutations was constructed and characterized. The mutant phi29 DNA polymerase can perform WGA at elevated temperatures (40-42°C), generate two to five times more of DNA amplification products, and has significantly increased half-life at 30 and 40°C, both in the presence or the absence of DNA substrate.

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

What Makes Y Family Pols Potential Candidates for Molecular Targeted Therapies and Novel Biotechnological Applications

Nature has evolved DNA polymerases (Pols) with different replication fidelity with the purpose of maintaining and faithfully propagating the genetic information. Besides the four classical Pols (Pol α, δ, ε, γ), mammalian cells contain at least twelve specialized Pols whose functions have been discovered recently and are still not completely elucidated. Among them, Pols belonging to the Y family contribute to cell survival by promoting DNA damage tolerance. They are primarily involved in the translesion synthesis (TLS) pathway, incorporating dNTPs in an error-free or error-prone manner, depending on the nature of the DNA lesion. From an evolutionary point of view, their high mutagenic potential seems to guarantee the proper flexibility of vital importance for both adaptation to a changeable environment and evolution of the species. These Pols are subjected to a complex network of regulation, since their uncontrolled access to DNA might promote mutagenesis and neoplastic transformation. Altered expression of Y family is a hallmark of several tumor types. In recent years, the unique structure and properties of Y family Pols have been exploited to design molecules that selectively interfere with the Pol of interest with minimal effect on normal cells. In addition, their distinctive properties have been applied to innovative techniques, such as compartmentalized self-replication (CSR), short-patch CSR, phage display and molecular breeding. These approaches are based on mutant Pols provided with novel and ameliorated features and find applications in various fields, from biotechnology to diagnostics, paleontology and forensic analysis.

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide -phosphate derivative

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

Evolving a polymerase for hydrophobic base analogues.

Hydrophobic base analogues (HBAs) have shown great promise for the expansion of the chemical and coding potential of nucleic acids but are generally poor polymerase substrates. While extensive synthetic efforts have yielded examples of HBAs with favorable substrate properties, their discovery has remained challenging. Here we describe a complementary strategy for improving HBA substrate properties by directed evolution of a dedicated polymerase using compartmentalized self-replication (CSR) with the archetypal HBA 5-nitroindole (d5NI) and its derivative 5-nitroindole-3-carboxamide (d5NIC) as selection substrates. Starting from a repertoire of chimeric polymerases generated by molecular breeding of DNA polymerase genes from the genus Thermus, we isolated a polymerase (5D4) with a generically enhanced ability to utilize HBAs. The selected polymerase. 5D4 was able to form and extend d5NI and d5NIC (d5NI(C)) self-pairs as well as d5NI(C) heteropairs with all four bases with efficiencies approaching, or exceeding, those of the cognate Watson-Crick pairs, despite significant distortions caused by the intercalation of the d5NI(C) heterocycles into the opposing strand base stack, as shown by nuclear magnetic resonance spectroscopy (NMR). Unlike Taq polymerase, 5D4 was also able to extend HBA pairs such as Pyrene: varphi (abasic site), d5NI: varphi, and isocarbostyril (ICS): 7-azaindole (7AI), allowed bypass of a chemically diverse spectrum of HBAs, and enabled PCR amplification with primers comprising multiple d5NI(C)-substitutions, while maintaining high levels of catalytic activity and fidelity. The selected polymerase 5D4 promises to expand the range of nucleobase analogues amenable to replication and should find numerous applications, including the synthesis and replication of nucleic acid polymers with expanded chemical and functional diversity.

Directed evolution of polymerase function by compartmentalized self-replication

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.