Synthesis and Applications of PNA and Modified PNA in Nanobiotechnology

The structural properties that enable DNA to serve as genetic material has also been used for many nanobiotechnological applications providing DNA-based nanomaterials and nanodevices. Peptide Nucleic Acids (PNAs) are DNA mimics in which the sugar-phosphate backbone has been replaced by a polyamide chain composed of N-(2-aminoethyl)glycine repeating units, covalently linked to nucleobases through a methylene carbonyl spacer. Compared to DNA, PNA can form duplexes more stable and are more sequence-selective. In this work, we investigated the use of PNA as tool for nanobiotechnology. PNA beacons were used in connection with HPLC, as biosensors for selective label-free detection of single mismatch in DNA. A PNA beacon containing a chiral monomer has also been synthesized and has shown an improvement both in binding and in fluorescence properties if compared to the corresponding achiral probe. Furthermore, we investigated the use of PNA as model for tunable materials througth which chirality can be amplified.Then, we studied the propagation of helicity induced by a C-terminal amino acid through PNA:PNA duplexes taking into account the effect of the amino acid side chain and charge, the solvent and temperature effects. Finally, we demonstrated that PNA can be used as powerful tools for molecular computers taking advantage of their higher selectivity and the higher stability of PNA:PNA duplexes if compared to DNA:DNA duplexes

Supporting Data for the Characterization of PNA-DNA Four-Way Junctions

Holliday or DNA four-way junctions (4WJs) are cruciform/bent structures composed of four DNA duplexes. 4WJs are key intermediates in homologous genetic recombination and double-strand break repair. To investigate 4WJs in vitro, junctions are assembled using four asymmetric DNA strands. The presence of four asymmetric strands about the junction branch point eliminates branch migration, and effectively immobilizes the resulting 4WJ. The purpose of these experiments is to show that immobile 4WJs composed of DNA and peptide nucleic acids (PNAs) can be distinguished from contaminating labile nucleic acid structures. These data compare the electrophoretic mobility of hybrid PNA–DNA junctions vs. i) a classic immobile DNA 4WJ, J1 and ii) contaminating nucleic acid structures

Interaction of HMG Proteins and H1 with Hybrid PNA-DNA Junctions

The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein‐binding surface of an immobilized four‐way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ‐PNA1 and 4WJ‐PNA3). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ\u27s. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two‐state equilibrium between: (i) an open‐x state favored by absence of Mg2+, low salt, and protein binding, and (ii) a compact stacked‐x state favored by Mg2+. 4WJ‐PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg+2, while 4WJ‐PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ‐PNA3 with nearly identical affinity (KDs) and 4WJ‐PNA1 with two‐fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs

Structure–Activity Relationship Assessment of Sophorolipid Ester Derivatives against Model Bacteria Strains

Sophorolipids (SLs) are glycolipids that consist of a hydrophilic sophorose head group covalently linked to a hydrophobic fatty acid tail. They are produced by fermentation of non-pathogenic yeasts such as Candida Bombicola. The fermentation products predominantly consist of the diacetylated lactonic form that coexists with the open-chain acidic form. A systematic series of modified SLs were prepared by ring opening of natural lactonic SL with n-alkanols of varying chain length under alkaline conditions and lipase-selective acetylation of sophorose primary hydroxyl groups. The antimicrobial activity of modified SLs against Gram-positive human pathogens was a function of the n-alkanol length, as well as the degree of sophorose acetylation at the primary hydroxyl sites. Modified SLs were identified with promising antimicrobial activities against Gram-positive human pathogens with moderate selectivity (therapeutic index, TI = EC50/MICB. cereus = 6–33). SL-butyl ester exhibited the best antimicrobial activity (MIC = 12 μM) and selectivity (TI = 33) among all SLs tested. Kinetic studies revealed that SL-ester derivatives kill B. cereus in a time-dependent manner resulting in greater than a 3-log reduction in cell number within 1 h at 2×MIC. In contrast, lactonic SL required 3 h to achieve the same efficiency

Characterization of the Structural and Protein Recognition Properties of Hybrid PNA-DNA Four-Way Junctions

The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA–DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg+2. For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg+2. The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b

Supporting data for the characterization of PNA–DNA four-way junctions

Holliday or DNA four-way junctions (4WJs) are cruciform/bent structures composed of four DNA duplexes. 4WJs are key intermediates in homologous genetic recombination and double-strand break repair. To investigate 4WJs in vitro, junctions are assembled using four asymmetric DNA strands. The presence of four asymmetric strands about the junction branch point eliminates branch migration, and effectively immobilizes the resulting 4WJ. The purpose of these experiments is to show that immobile 4WJs composed of DNA and peptide nucleic acids (PNAs) can be distinguished from contaminating labile nucleic acid structures. These data compare the electrophoretic mobility of hybrid PNA–DNA junctions vs. i) a classic immobile DNA 4WJ, J1 and ii) contaminating nucleic acid structures

The Chemoenzymatic Synthesis of Glycan-Terminated Oligo(Leu)x

Synthesis with papain for peptides with an N-terminal glucose group indicated a preference for the grafters with a longer linker between the glucose moiety and the Phe. Computational modeling was performed to investigate further, and here are all the files used to generate the models as well as the resulting models

Peptide Modified Electrospun Glycopolymer Fibers

Oligo(Glu70-co-Leu30), a peptide synthesized by protease catalysis, is functionalized at the N-terminus with a 4-pentenoyl unit and grafted to polyLSL[6\u2032Ac,6\u2033Ac], a glycopolymer prepared by ring-opening metathesis polymerization of lactonic sophorolipid diacetate. First, polyLSL[6'Ac,6\u201dAc] fiber mats are fabricated by electrospinning. Oxidation of the fiber mats and subsequent reaction with cysteamine lead to thiol-functionalized fiber mats with no significant morphology changes. Grafting of the alkene-modified oligopeptide to thiol-functionalized polyLSL[6\u2032Ac,6\u2033Ac] fiber mats is achieved via \u201cthiol-ene\u201d click reaction. X-ray photoelectron spectroscopy analysis to characterize peptide grafting reveals that about 50 mol% of polyLSL[6\u2032Ac,6\u2032\u2032Ac] repeat units at fiber surfaces are decorated with a peptide moiety, out of which about 1/3 of the oligo(Glu70-co-Leu30) units are physically adsorbed to polyLSL[6\u2032Ac,6\u2032\u2032Ac]. The results of this work pave the way to precise engineering of polyLSL fiber mats that can be decorated with a potentially wide range of molecules that tailor surface chemistry and biological properties. (Figure presented.)

DataSheet1_The chemoenzymatic synthesis of glycan-terminated oligo(Leu)x.docx

Introduction: Glycopeptides contain carbohydrate moieties (glycans) covalently attached to the side chain and/or terminal peptide units. Since glycans are present on cell surfaces, these constructs can potentially address a wide array of therapeutic functions. To overcome the deficiencies associated with current synthetic routes to glycopeptides, such as costly processes and toxic reagents, this work aimed to develop versatile environmentally friendly protease-catalyzed peptide synthesis routes to peptides decorated with a glycan at their N-terminus.Methods: “Grafters” were first synthesized that consist of a glycan conjugated directly, or through a spacer, to the amine group of L-Phe-ethyl ester (Phe-OEt). The role of Phe-OEt is to increase the conjugate’s recognition by the protease (papain) catalytic active site. A series of grafters were synthesized with variation of the glycan structure, linkage-chemistry, and presence of an oligo (ethylene glycol) “spacer” of varied length between the glycan and Phe-OEt moiety. High grafter efficiency will result by the successful acceptance of the grafter at the enzymes S1/S2 subsites, formation of an acyl enzyme complex and subsequent conversion to glycan-terminated oligo(Leu)x (x ≥ 1), as opposed to construction of non-glycan N-terminated oligo(Leu)x.Results and discussion: While glycan-Phe-OEt grafters without a spacer between the glycan and Phe-OEt resulted in low grafter efficiency (8.3% ± 2.0%), insertion of a short oligo (ethylene glycol) spacer between the glycan and Phe-OEt moieties (glycan-PEGn-Phe-OEt, n ≥ 3) increased the grafter efficiency by 3-fold–24.5% ± 1.8%. In addition, computational modeling was performed using Rosetta software provided insights on a molecular level of how grafter efficiency is influenced by the PEG spacer length.</p

Sophorolipid Butyl Ester Diacetate Does Not Affect Macrophage Polarization but Enhances Astrocytic Glial Fibrillary Acidic Protein Expression at Micromolar Concentrations in Vitro

Peritoneal macrophages (PMACs) and spinal cord astrocytes were exposed to varying concentrations of soluble sophorolipid butyl ester diacetate (SLBEDA) in vitro. Macrophages and astrocytes demonstrated no decrease in viability in response to SLBEDA. Studying pro- and anti-inflammatory genes, PMACs did not show a shift toward a pro-inflammatory phenotype. However, at higher concentrations (3 and 30 μM), astrocytes showed an increase in their expression of glial acidic fibrillary protein. This novel category of compounds poses low risk to PMAC and astrocyte viability; however, the effect on PMAC polarization and astrocyte reactivity requires more elucidation