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Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.

The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

Synthetic genetics is a subdiscipline of synthetic biology that aims to develop artificial genetic polymers (also referred to as xeno-nucleic acids or XNAs) that can replicate in vitro and eventually in model cellular organisms. This field of science combines organic chemistry with polymerase engineering to create alternative forms of DNA that can store genetic information and evolve in response to external stimuli. Practitioners of synthetic genetics postulate that XNA could be used to safeguard synthetic biology organisms by storing genetic information in orthogonal chromosomes. XNA polymers are also under active investigation as a source of nuclease resistant affinity reagents (aptamers) and catalysts (xenozymes) with practical applications in disease diagnosis and treatment. In this review, we provide a structural perspective on known antiparallel duplex structures in which at least one strand of the Watson-Crick duplex is composed entirely of XNA. Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available. Given the growing interest in xenobiology projects, we chose to compare the structural features of XNA polymers and discuss their potential to access new regions of nucleic acid fold space.

Squaliforme sharks are a common but relatively vulnerable bycatch in many deep water fisheries. Eleven species of squaliforme shark are commonly caught at depths of 200–1200 m on Chatham Rise, New Zealand, and their diversity suggests they might occupy different niches. The diets of 133 Deania calcea and 295 Squalus acanthias were determined from examination of stomach contents. The diet of D. calcea was characterised by mesopelagic fishes, and S. acanthias by benthic to pelagic fishes, but was more adaptive and included likely scavenging. Multivariate analyses found the most important predictors of diet variability in S. acanthias were year, bottom temperature, longitude, and fish weight. The diet of the nine other commonly caught squaliforme sharks was reviewed, and the spatial and depth distribution of all species on Chatham Rise described from research bottom trawl survey catches. The eleven species had a variety of different diets, and depth and location preferences, consistent with niche separation to reduce interspecific competition. Four trophic groups were identified, characterised by: mesopelagic fishes and invertebrates (Centroselachus crepidater, D. calcea, and Etmopterus lucifer); mesopelagic and benthopelagic fishes and invertebrates (Centroscymnus owstoni, Etmopterus baxteri); demersal and benthic fishes (Centrophorus squamosus, Dalatias licha, Proscymnodon plunketi); and a generalist diet of fishes and invertebrates (S. acanthias). The trophic levels of the species in each of the four groups were estimated as 4.18–4.24, 4.20–4.23, 4.24–4.48, and 3.84 respectively. The diet of Oxynotus bruniensis and Squalus griffini are unknown. The different niches occupied by different species are likely to influence their vulnerability to bottom trawl fisheries. Some species may benefit from fisheries through an increased availability of scavenged prey. [ABSTRACT FROM AUTHOR]

Squaliforme sharks are a common but relatively vulnerable bycatch in many deep water fisheries. Eleven species of squaliforme shark are commonly caught at depths of 200–1200 m on Chatham Rise, New Zealand, and their diversity suggests they might occupy different niches. The diets of 133 Deania calcea and 295 Squalus acanthias were determined from examination of stomach contents. The diet of D. calcea was characterised by mesopelagic fishes, and S. acanthias by benthic to pelagic fishes, but was more adaptive and included likely scavenging. Multivariate analyses found the most important predictors of diet variability in S. acanthias were year, bottom temperature, longitude, and fish weight. The diet of the nine other commonly caught squaliforme sharks was reviewed, and the spatial and depth distribution of all species on Chatham Rise described from research bottom trawl survey catches. The eleven species had a variety of different diets, and depth and location preferences, consistent with niche separation to reduce interspecific competition. Four trophic groups were identified, characterised by: mesopelagic fishes and invertebrates (Centroselachus crepidater, D. calcea, and Etmopterus lucifer); mesopelagic and benthopelagic fishes and invertebrates (Centroscymnus owstoni, Etmopterus baxteri); demersal and benthic fishes (Centrophorus squamosus, Dalatias licha, Proscymnodon plunketi); and a generalist diet of fishes and invertebrates (S. acanthias). The trophic levels of the species in each of the four groups were estimated as 4.18–4.24, 4.20–4.23, 4.24–4.48, and 3.84 respectively. The diet of Oxynotus bruniensis and Squalus griffini are unknown. The different niches occupied by different species are likely to influence their vulnerability to bottom trawl fisheries. Some species may benefit from fisheries through an increased availability of scavenged prey. [ABSTRACT FROM AUTHOR]