Construction of a Holliday Junction in Small Circular DNA Molecules for Stable Motifs and Two‐Dimensional Lattices

Design rules for DNA nanotechnology have been mostly learnt from using linear single‐stranded (ss) DNA as the source material. For example, the core structure of a typical DAO (double crossover, antiparallel, odd half‐turns) tile for assembling 2D lattices is constructed from only two linear ss‐oligonucleotide scaffold strands, similar to two ropes making a square knot. Herein, a new type of coupled DAO (cDAO) tile and 2D lattices of small circular ss‐oligonucleotides as scaffold strands and linear ss‐oligonucleotides as staple strands are reported. A cDAO tile of cDAO‐c64nt (c64nt: circular 64 nucleotides), shaped as a solid parallelogram, is constructed with a Holliday junction (HJ) at the center and two HJs at both poles of a c64nt; similarly, cDAO‐c84nt, shaped as a crossed quadrilateral composed of two congruent triangles, is formed with a HJ at the center and four three‐way junctions at the corners of a c84nt. Perfect 2D lattices were assembled from cDAO tiles: infinite nanostructures of nanoribbons, nanotubes, and nanorings, and finite nanostructures. The structural relationship between the visible lattices imaged by AFM and the corresponding invisible secondary and tertiary molecular structures of HJs, inclination angle of hydrogen bonds against the double‐helix axis, and the chirality of the tile can be interpreted very well. This work could shed new light on DNA nanotechnology with unique circular tiles.     

Synthesis of C8‐N‐Arylamine‐Modified 2′‐Deoxyguanosine‐5′‐Triphosphates and Their Effects on Primer Extension by DNA Polymerases

C8‐N‐arylamine adducts of 2′‐deoxyguanosine (2′‐dG) play an important role in the induction of the chemical carcinogenesis caused by aromatic amines. C8‐N‐acetyl‐N‐arylamine dG adducts that differ in their substitution pattern in the aniline moiety were converted by cycloSal technology into the corresponding C8‐N‐acetyl‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates and C8‐NH‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates. Their conformation preference has been investigated by NOE spectroscopy and DFT calculations. The substrate properties of the C8‐dG adducts were studied in primer‐extension assays by using Klenow fragment exo^? of Escherichia coli DNA polymerase I and human DNA polymerase β. It was shown that the incorporation was independent of the substitution pattern in the aryl moiety and the N‐acetyl group. Although the triphosphates were poor substrates for the human polymerases, they were incorporated twice before the termination of the elongation process occurred; this might demonstrate the importance of C8‐N‐arylamine‐2′‐deoxyguanosine‐5′‐triphosphates in chemical carcinogenesis.     

One‐Step Cationic Poly(D,L‐lactic acid) Particle Synthesis by Diafiltration and Interactions with Plasmid DNA

Summary: Diafiltration was used for the one step preparation of cationic poly(D ,L ‐lactic acid) particles in the presence of poly(ethylenimine) (PEI). First, parameters impacting the formation of plain PLA particles were investigated: the chemical nature of the solvent and the polymer concentration within the organic phase. Then, PEI was added to the organic solution and this mixture was subsequently dialyzed against water. The molecular architecture of the polycation was essential and colloidal particles were only obtained with a branched PEI of 25 000 g · mol⁻¹. Increasing the PEI concentration in the mixture led to a particle mean size reduction and a zeta‐potential increase. In any case, a maximum of 8% of the initial PEI input was recovered at the interface. Finally, the study of the interactions of these colloids with DNA showed that the amount of DNA bound at the saturation plateau increased with increasing inputs of PEI in the formulation.

SEM micrograph of cationic poly(D ,L ‐lactic acid) particles.     

Optimization of an Electrochemical DNA Assay by Using a 48‐Electrode Array and Redox Amplification Studies by Means of Scanning Electrochemical Microscopy

Sensible DNA : An electrochemical DNA assay based on specific Salmonella spp. capture probes and enzyme labeling with alkaline phosphatase was optimized by using a 48‐electrode microarray and scanning electrochemical microscopy (SECM). SECM was further used to evaluate potential amplification strategies due to redox cycling.

     

DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells

A deoxyadenosine triphosphate (dATP) analogue for DNA labeling was synthesized with the 1‐methylcyclopropene (1MCP) group at the 7‐position of 7‐deaza‐2′‐deoxyadenosine and applied for primer extension experiments. The real‐time kinetic data reveals that this 1MCP‐modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP‐modified deoxyuridine triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site‐specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.     

Structural Insights into the Recognition of N2‐Aryl‐ and C8‐Aryl DNA Lesions by the Repair Protein XPA/Rad14

Aromatic amines are strongly carcinogenic. They are activated in the liver to give reactive nitrenium ions that react with nucleobases within the DNA duplex. The reaction occurs predominantly at the C8 position of the dG base, thereby giving C8‐acetyl‐aryl‐ or C8‐aryl‐dG adducts in an electrophilic aromatic substitution reaction. Alternatively, reaction with the exocyclic 2‐NH₂ group is observed. Although the C8 adducts retain base‐pairing properties, base pairing is strongly compromised in the case of the N² adducts. Here we show crystal structures of two DNA lesions, N²‐acetylnaphthyl‐dG and C8‐fluorenyl‐dG, within a DNA duplex recognized by the repair protein Rad14. The structures confirm that two molecules of the repair protein recognize the lesion and induce a 72 or 78° kink at the site of the damage. Importantly, the same overall kinked structure is induced by binding of the repair proteins, although the structurally different lesions result in distinct stacking interactions of the lesions within the duplex. The results suggest that the repair protein XPA/Rad14 is a sensor that recognizes flexibility. The protein converts the information that structurally different lesions are present in the duplex into a unifying sharply kinked recognition motif.     

Translational Diffusion and Interaction of a Photoreceptor and Its Cognate Transducer Observed in Giant Unilamellar Vesicles by Using Dual‐Focus FCS

In order to monitor membrane–protein binding in lipid bilayers at physiological protein concentrations, we employed the recently developed dual‐focus fluorescence correlation spectroscopy (2fFCS) technique. In a case study on a photoreceptor consisting of seven transmembrane helices and its cognate transducer (two transmembrane helices), the lateral diffusion for these integral membrane proteins was analyzed in giant unilamellar vesicles (GUVs). The two‐dimensional diffusion coefficients of both separately diffusing proteins differ significantly, with D=2.2×10^?8 cm² s^?1 for the photoreceptor and with D=4.1×10^?8 cm² s^?1 for the transducer. In GUVs with both membrane proteins present together, we observed significantly smaller diffusion coefficients for labelled transducer molecules; this indicates the presence of larger diffusing units and therefore intermolecular protein binding. Based on the phenomenological dependence of diffusion coefficients on the molecule's cylindrical radius, we are able to estimate the degree of membrane protein binding on a quantitative level.     

Strictly Conserved Residues in Euphorbia tirucalli β‐Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation–π Interaction and CH–π Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli β‐amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5 % of the wild‐type activity for the W612H variant, 43 % for W612F, and 63 % for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in π‐electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation–π interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E‐ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56–78 % of wild‐type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67 %), which indicates that the π electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH–π interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation–π interaction.     

Stereochemical elucidation of complex α‐methyl and β‐methylene carbon resonances in poly(2‐hydroxy ethyl methacrylate‐co‐methacrylonitrile) by 2D NMR

Copolymers of 2‐hydroxy ethyl methacrylate and methacrylonitrile (H/M) of different composition were synthesized by free radical bulk polymerization using azobisisobutyronitrile (AIBN) as an initiator under nitrogen atmosphere. The copolymers composition were calculated from ¹H and quantitative ¹³C{¹H}NMR spectra. The complete spectral assignment of complex and overlapped α‐methyl and β‐methylene carbon regions in ¹³C{¹H} NMR spectrum in term of compositional and configurational sequences of H/M copolymers were done with the help of two‐dimensional heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY). © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

The Binomial “Inflammation-Epigenetics” in Breast Cancer Progression and Bone Metastasis: IL-1β Actions Are Influenced by TET Inhibitor in MCF-7 Cell Line

The existence of a tight relationship between inflammation and epigenetics that in primary breast tumor cells can lead to tumor progression and the formation of bone metastases was investigated. It was highlighted how the induction of tumor progression and bone metastasis by Interleukin-1 beta, in a non-metastatic breast cancer cell line, MCF-7, was dependent on the de-methylating actions of ten-eleven translocation proteins (TETs). In fact, the inhibition of their activity by the Bobcat339 molecule, an inhibitor of TET enzymes, determined on the one hand, the modulation of the epithelial-mesenchymal transition process, and on the other hand, the reduction in the expression of markers of bone metastasis, indicating that the epigenetic action of TETs is a prerequisite for IL-1β-dependent tumor progression and bone metastasis formation.     

Solvothermal Synthesis,Crystal Structure and DNA Binding Studies of a 3D Supramolecular Complex {[Cu(phen)CN][Cu(phen)][Cu(CN)<Subscript>2</Subscript>]}<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> Assembled by Double Curvy Chains

A new cyano-bridged copper (I) complex {[Cu(phen)CN][Cu(phen)][Cu(CN)₂]} _n (1) (phen = 1,10-phenanthroline) was synthesized under the solvothermal conditions, and characterized by elemental analysis, FI-IR spectra and single-crystal X-ray diffraction. The structure analysis reveals that complex 1 contains two [Cu(phen)CN][Cu(phen)][Cu(CN)₂] subunits, being, respectively, constructed into two chains with different cyano backbones. The resulting chains offer a double curvy chain by Cu···Cu weak interactions and π–π stacking interactions. It is exciting that the double curvy chains adopt anti-parallel array and result in a novel 3D architecture through π–π stacking interactions. According to the supramolecular self-assembly of complex 1, four types of stacking mode of phenanthroline moieties are given and discussed. Absorption and fluorescence titration studies of complex 1 with calf thymus DNA are suggestive of the intercalation binding mode with a intrinsic binding constant of 7.52 × 10³ M⁻¹ and a linear Stern-Volmer quenching constant of 1.02 × 10⁵ M⁻¹.