John C. Chaput

The structural diversity of artificial genetic polymers

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 Currently, only a handful of XNA structures have been archived in the Protein Data Bank as compared to the more than 100 000 structures that are now available.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.

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase.

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 enzymes 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.

Synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue

Abstract Threose nucleic acid (TNA) is an artificial genetic polymer capable of undergoing Darwinian evolution to produce aptamers with affinity to specific targets. This property, coupled with a backbone structure that is refractory to nuclease digestion, makes TNA an attractive biopolymer system for diagnostic and therapeutic applications. Expanding the chemical diversity of TNA beyond the natural bases would enable the development of functional TNA molecules with enhanced physiochemical properties. Here, we describe the synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue (1,3-diaza-2-oxo-phenothiazine, tCfTP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer-extension assays reveal that tCfTP is efficiently added to the growing end of a TNA primer. Detailed kinetic assays indicate that tCfTP and tCTP have comparable rates for the first nucleotide incorporation step (kobs1). However, addition of the second nucleotide (kobs2) is 700-fold faster for tCfTP than tCTP due the increased effects of base stacking. Last, we found that TNA replication using tCfTP in place of tCTP exhibits 98.4% overall fidelity for the combined process of TNA transcription and reverse transcription. Together, these results expand the chemical diversity of enzymatically generated TNA molecules to include a hydrophobic base analogue with strong fluorescent properties that is compatible with in vitro selection.

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.

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four‐carbon threose backbone in place of the five‐carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson–Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base‐pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A‐like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric “breathing” fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.

A Parallel Stranded G-Quadruplex Composed of Threose Nucleic Acid (TNA).

G‐rich sequences can adopt four‐stranded helical structures, called G‐quadruplexes, that self‐assemble around monovalent cations like sodium (Na+) and potassium (K+). Whether similar structures can be formed from xeno‐nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α‐l‐threofuranosyl nucleic acid) to adopt a four‐stranded helical structure based on a planar G‐quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution‐state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self‐assemble into stable G‐quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G‐quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self‐assembling into stable G‐quadruplex structures.

RNA World: Visualizing primer extension without enzymes

X-ray crystallography has been used to observe the synthesis of RNA in the absence of enzymes with atomic resolution.

Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

Activation of innate immune responses by a CpG oligonucleotide sequence composed entirely of threose nucleic acid.

Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs). Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base-pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. Here, we evaluated a TNA oligonucleotide derived from a CpG oligonucleotide sequence known to activate TLR9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.

Made in translation

Evolution of highly functionalized DNA could enable the discovery of artificial nucleic acid sequences with different properties to natural DNA. Now, an artificial translation system has been designed that can support the evolution of non-natural sequence-defined nucleic acid polymers carrying eight different functional groups on 32 codons.