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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)

   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. Nanopores map the acid-base properties of a single site in a single DNA molecule

   https://doi.org/10.1093/nar/gkae518  

   Smith, Drew C. ; Thomas, Christopher A. ; Craig, Jonathan M. ; Brinkerhoff, Henry ; Abell, Sarah J. ; Franzi, Michaela C. ; Carrasco, Jessica D. ; Hoshika, Shuichi ; Benner, Steven A. ; Gundlach, Jens H. ; et al ( June 2024 , Nucleic Acids Research)

   Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino acids are known to have pKas near neutral pHs at which nanopore sequencing is typically performed. Previous reports have suggested that the nanopore signal may be sensitive to the protonation state of an individual moiety. We sequenced ion currents with the MspA nanopore using a single stranded DNA containing a single non-canonical DNA base (Z) at various pH conditions. The Z-base has a near-neutral pKa ∼ 7.8. We find that the measured ion current is remarkably sensitive to the protonation state of the Z-base. We demonstrate how nanopores can be used to localize and determine the pKa of individual moieties along a polymer. More broadly, these experiments provide a path to mapping different protonation sites along polymers and give insight in how to optimize sequencing of polymers that contain moieties with near-neutral pKas.

    

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3. Abiotic stressors impact outer membrane vesicle composition in a beneficial rhizobacterium: Raman spectroscopy characterization

   https://doi.org/10.1038/s41598-020-78357-4  

   Potter, Matthew ; Hanson, Cynthia ; Anderson, Anne J. ; Vargis, Elizabeth ; Britt, David W. ( December 2020 , Scientific Reports)

   Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacteriumPseudomonas chlororaphisO6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H₂O₂-released from roots during plant stress responses. Atomic force microscopy revealed 40–300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles ofPcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids thanPcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H₂O₂-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.

    

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4. A high-performance polymer composite column for coronavirus nucleic acid purification

   https://doi.org/10.1038/s41598-024-51671-x  

   Zarouri, Akli ; Barnes, Aaron_M T ; Aboubakr, Hamada ; Thekkudan_Novi, Vinni ; Dong, Qiuchen ; Nelson, Andrew ; Goyal, Sagar ; Abbas, Abdennour ( December 2024 , Scientific Reports)

   Here, we report the development of a novel polymer composite (PC) purification column and kit. The performance of the PC columns was compared to conventional silica gel (SG) columns for the purification of nucleic acids from coronaviruses, including SARS-CoV-2, in 82 clinical samples. The results shows that PC-based purification outperforms silica gel (SG)-based purification by enabling a higher sensitivity (94%), accuracy (97%), and by eliminating false positives (100% specificity). The high specificity is critical for efficient patient triage and resource management during pandemics. Furthermore, PC-based purification exhibits three times higher analytical precision than a commonly used SG-based nucleic acid purification thereby enabling a more accurate quantification of viral loads and higher reproducibility.

    

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5. Head on Comparison of Self‐ and Nano‐Assemblies of Gamma Peptide Nucleic Acid Amphiphiles

   https://doi.org/10.1002/adfm.202109552  

   Malik, Shipra ; Kumar, Vikas ; Liu, Chung‐Hao ; Shih, Kuo‐Chih ; Krueger, Susan ; Nieh, Mu‐Ping ; Bahal, Raman ( November 2021 , Advanced Functional Materials)

   Peptide nucleic acids (PNAs) are nucleic acid analogs with hybridization properties and enzymatic stability superior to that of DNA. In addition to gene targeting applications, PNAs have garnered significant attention as bio‐polymers due to the Watson–Crick‐based molecular recognition and flexibility of synthesis. Here, PNA amphiphiles are engineered using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γ  PNA) amphiphiles self‐assemble into spherical vesicles. Further, nano‐assemblies (NA) are formulated using the amphiphilic γ  PNA as a polymer via ethanol injection‐based protocols. Comprehensive head‐on comparison of the physicochemical and cellular uptake properties of PNA derived self‐ and NA is performed. Small‐angle neutron and X‐ray scattering analysis reveal ellipsoidal morphology of γ  PNA NA that results in superior cellular delivery compate to the spherical self‐assembly. Next, the functional activities of γ  PNA self‐and NA in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis‐based assays are compared. Overall, it is established that γ  PNA amphiphile is a functionally active bio‐polymer to formulate NA for a wide range of biomedical applications.

    

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