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1. Crystallographic analysis of engineered polymerases synthesizing phosphonomethylthreosyl nucleic acid

   https://doi.org/10.1093/nar/gkac792  

   Hajjar, Mohammad; Chim, Nicholas; Liu, Chao; Herdewijn, Piet; Chaput, John C. (September 2022, Nucleic Acids Research)

   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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2. Stability and mechanism of threose nucleic acid toward acid-mediated degradation

   https://doi.org/10.1093/nar/gkad716  

   Lee, Erica M.; Setterholm, Noah A.; Hajjar, Mohammad; Barpuzary, Bhawna; Chaput, John C. (August 2023, Nucleic Acids Research)

   Abstract Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2′-phosphodiester linkage. Similar results observed for 2′,5′-linked DNA and 2′-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by β-elimination of the 2′-phosphodiester linkage to produce an upstream cleavage product with a 2′-threose sugar and a downstream cleavage product with a 3′ terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications. 

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3. Transliteration of synthetic genetic enzymes

   https://doi.org/10.1093/nar/gkab923  

   Wang, Yajun; Liu, Xiaolin; Shehabat, Mouhamad; Chim, Nicholas; Chaput, John C. (October 2021, Nucleic Acids Research)

   Abstract Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding. 

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4. Design, optimization and analysis of large DNA and RNA nanostructures through interactive visualization, editing and molecular simulation

   https://doi.org/10.1093/nar/gkaa417  

   Poppleton, Erik; Bohlin, Joakim; Matthies, Michael; Sharma, Shuchi; Zhang, Fei; Šulc, Petr (May 2020, Nucleic Acids Research)

   Abstract This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to design and analysis of a range of DNA/RNA nanostructures. 

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5. Synthesis and polymerase recognition of a pyrrolocytidine TNA triphosphate

   https://doi.org/10.1002/bip.23388  

   Mei, Hui; Wang, Yajun; Yik, Eric J.; Chaput, John C. (July 2020, Biopolymers)

   Abstract 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, tC^pTP) that maintains Watson‐Crick base pairing with guanine. Polymerase‐mediated primer extension assays show that tC^pTP is an efficient substrate for Kod‐RI, a DNA‐dependent TNA polymerase developed to explore the functional properties of TNA byin vitroselection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tC^pTP and 7‐deaza‐tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available forin vitroselection. 

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