Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno‐nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α‐L‐Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6‐phenyl‐pyrrolocytosine, tCpTP) that maintains Watson‐Crick base pairing with guanine. Polymerase‐mediated primer extension assays show that tCpTP is an efficient substrate for Kod‐RI, a DNA‐dependent TNA polymerase developed to explore the functional properties of TNA by
Mutant polymerases capable of 2′ fluoro-modified nucleic acid synthesis and amplification with improved accuracy
Nonnatural nucleic acids (xeno nucleic acids, XNA) can possess several useful properties such as expanded reactivity and nuclease resistance, which can enhance the utility of DNA as a biotechnological tool. Native DNA polymerases are unable to synthesize XNA, so, in recent years mutant XNA polymerases have been engineered with sufficient activity for use in processes such as PCR. While substantial improvements have been made, accuracy still needs to be increased by orders of magnitude to approach natural error rates and make XNA polymerases useful for applications that require high fidelity. Here, we systematically evaluate leading Taq DNA polymerase mutants for their fidelity during synthesis of 2′F XNA. To further improve their accuracy, we add mutations that have been shown to increase the fidelity of wild-type Taq polymerases, to some of the best current XNA polymerases (SFM4–3, SFM4–6, and SFP1). The resulting polymerases show significant improvements in synthesis accuracy. In addition to generating more accurate XNA polymerases, this study also informs future polymerase engineering efforts by demonstrating that mutations that improve the accuracy of DNA synthesis may also have utility in improving the accuracy of XNA synthesis.
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- Award ID(s):
- 1752924
- NSF-PAR ID:
- 10411367
- Date Published:
- Journal Name:
- RSC Chemical Biology
- Volume:
- 3
- Issue:
- 8
- ISSN:
- 2633-0679
- Page Range / eLocation ID:
- 1044 to 1051
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract in vitro selection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCpTP and 7‐deaza‐tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available forin vitro selection. -
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Abstract Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3′-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson–Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.
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null (Ed.)Abstract The helical structures of DNA and RNA were originally revealed by experimental data. Likewise, the development of programs for modeling these natural polymers was guided by known structures. These nucleic acid polymers represent only two members of a potentially vast class of polymers with similar structural features, but that differ from DNA and RNA in the backbone or nucleobases. Xeno nucleic acids (XNAs) incorporate alternative backbones that affect the conformational, chemical, and thermodynamic properties of XNAs. Given the vast chemical space of possible XNAs, computational modeling of alternative nucleic acids can accelerate the search for plausible nucleic acid analogs and guide their rational design. Additionally, a tool for the modeling of nucleic acids could help reveal what nucleic acid polymers may have existed before RNA in the early evolution of life. To aid the development of novel XNA polymers and the search for possible pre-RNA candidates, this article presents the proto-Nucleic Acid Builder (https://github.com/GT-NucleicAcids/pnab), an open-source program for modeling nucleic acid analogs with alternative backbones and nucleobases. The torsion-driven conformation search procedure implemented here predicts structures with good accuracy compared to experimental structures, and correctly demonstrates the correlation between the helical structure and the backbone conformation in DNA and RNA.more » « less
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Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth’s genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodeling functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further in vitro DNA amplification advancements.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d2cb00064d
