Four-arm Junctions Research Articles

Novel label-free fluorescence aptasensor for chloramphenicol detection based on a DNA four-arm junction-assisted signal amplification strategy

A novel label-free fluorescence aptasensor was established for chloramphenicol (CAP) detection by DNA four-arm junction-assisted target recycling and SYBR Green I dye-aided fluorescence-signal amplification. The CAP aptamer was hybridized to its complementary strand (primer) to form a double-stranded primer/aptamer complex. In the presence of CAP, aptamers can specifically bind with CAP to dissociate primers, which can trigger the self-assembly of four hairpins to continuously generate DNA four-arm junctions. After digesting the excess hairpins using T7 exonuclease, SYBR Green I was inserted into the base pair-rich DNA four-arm junctions, which led to a significant increase in fluorescence intensity. Under optimal conditions, the developed aptasensor can detect CAP in a linear range of 1.0 pg mL−1 to 10 ng mL−1 with a detection limit of 0.72 pg mL−1. The recovery rates in milk and honey ranged from 90.3% to 106.6%. Thus, the method shows substantial potential for CAP detection in food products.

Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

Design of the First Italian Roundabout with Jointed Plain Concrete Pavement

This work presents the results of the overall design of the first Italian roundabout with jointed plain concrete pavement. The examined case study complies with current international standards and practices for geometry of roundabouts and road pavements. The construction of a concrete pavement in an urban roundabout will better manage and slow down fast vehicular flows, and increase traffic fluidity in an important junction, trafficked by heavy vehicles, where maintenance works should be reduced to avoid queues. The design of the roundabout involved several competences for: defining the geometry of the four-arm junction, designing the thickness of the jointed plain concrete pavement both on the circular crown and the arms, studying the mix design of a high resistance concrete. As regard to the pavement, the result of the study was an un-dowelled concrete pavement composed of square slabs laid on a cement concrete subbase and a granular layer. The shape of the slabs has been designed to optimize the structural performance of their material, which is a high strength concrete mix derived from an extensive laboratory test work. In general, the results summarized approaches typical of different design conditions: urban ones for traffic flow and safety needs; high-traffic ones for the chosen pavement type; airport ones for the absence of dowel and tie bars at the joints. Indeed, the article has highlighted that the design process of a concrete roundabout requires multiple analyses to consider various features and correctly re-design an existing urban junction. Its geometrical design, the structural design of the concrete pavement and the theoretical and experimental design of the concrete mix were the main phases of this process and they needed different competences to conduct comprehensive and appropriate analyses.

Outlier Detection in Large-Scale Traffic Data by Naïve Bayes Method and Gaussian Mixture Model Method

It is meaningful to detect outliers in traffic data for traffic management. However, this is a massive task for people from largescale database to distinguish outliers. In this paper, we present two methods: Kernel Smoothing Naïve Bayes (NB) method and Gaussian Mixture Model (GMM) method to automatically detect any hardware errors as well as abnormal traffic events in traffic data collected at a four-arm junction in Hong Kong. Traffic data was recorded in a video format, and converted to spatial-temporal (ST) traffic signals by statistics. The ST signals are then projected to a two-dimensional (2D) (x, y)-coordinate plane by Principal Component Analysis (PCA) for dimension reduction. We assume that inlier data are normal distributed. As such, the NB and GMM methods are successfully applied in OD (Outlier Detection) for traffic data. The kernel smooth NB method assumes the existence of kernel distributions in traffic data and uses Bayes' Theorem to perform OD. In contrast, the GMM method believes the traffic data is formed by the mixture of Gaussian distributions and exploits confidence region for OD. This paper would address the modeling of each method and evaluate their respective performances. Experimental results show that the NB algorithm with Triangle kernel and GMM method achieve up to 93.78% and 94.50% accuracies, respectively.

DNA nanotubes assembled from tensegrity triangle tiles with circular DNA scaffolds.

Using small circular DNA molecules of different lengths as scaffolds, we successfully synthesised DNA nanotubes consisting of Mao's DNA tensegrity triangle tiles with four-arm junctions (Holliday junctions) at all vertices. Due to the intrinsic curvature of the triangle tile and the consecutive tile alignment, the 2D arrays are organised in the form of nanotubes. Two sized triangle tiles with equilateral side lengths of 1.5 and 2.5 full helical turns are connected by the sticky ended cohesion of a duplex with a length of 2.5 helical turns respectively, and their parallel lozenge tiling lattices were demonstrated by high resolution AFM images, where the former lozenge unit cell has a lattice constant of 13.6 nm, and the latter has a larger lattice constant of 17.0 nm. Modification of the triangle tile with infinitesimal disturbance on side lengths and insertion of one thymine single stranded loop at every vertex resulted in comparably similar nanotubes.

Design of Wireframe DNA Nanostructures-DNA Gridiron.

Self-assembling nucleic acid molecules have shown merit as versatile materials for organizing and constructing nanoscale structures with both 2D and 3D geometries. This chapter focuses on strategies in designing DNA gridiron nanostructures based on four-arm junction. This design strategies aims at controlling DNA self-assembly with a higher degree of spatial precision by depicting arbitrary 3D geometries with their wireframe outlines using DNA helices (for edges) and four-arm junctions (for vertices).

Construction and Structure Determination of a Three-Dimensional DNA Crystal.

Structural DNA nanotechnology combines branched DNA junctions with sticky-ended cohesion to create self-assembling macromolecular architectures. One of the key goals of structural DNA nanotechnology is to construct three-dimensional (3D) crystalline lattices. Here we present a new DNA motif and a strategy that has led to the assembly of a 3D lattice. We have determined the X-ray crystal structures of two related constructs to 3.1 Å resolution using bromine-derivatized crystals. The motif we used employs a five-nucleotide repeating sequence that weaves through a series of two-turn DNA duplexes. The duplexes are tied into a layered structure that is organized and dictated by a concert of four-arm junctions; these in turn assemble into continuous arrays facilitated by sequence-specific sticky-ended cohesion. The 3D X-ray structure of these DNA crystals holds promise for the design of new structural motifs to create programmable 3D DNA lattices with atomic spatial resolution. The two arrays differ by the use of four or six repeats of the five-nucleotide units in the repeating but statistically disordered central strand. In addition, we report a 2D rhombuslike array formed from similar components.

Coverage percentage and raman measurement of cross-tile and scaffold cross-tile based DNA nanostructures

We present two free-solution annealed DNA nanostructures consisting of either cross-tile CT1 or CT2. The proposed nanostructures exhibit two distinct structural morphologies, with one-dimensional (1D) nanotubes for CT1 and 2D nanolattices for CT2. When we perform mica-assisted growth annealing with CT1, a dramatic dimensional change occurs where the 1D nanotubes transform into 2D nanolattices due to the presence of the substrate. We assessed the coverage percentage of the 2D nanolattices grown on the mica substrate with CT1 and CT2 as a function of the concentration of the DNA monomer. Furthermore, we fabricated a scaffold cross-tile (SCT), which is a new design of a modified cross-tile that consists of four four-arm junctions with a square aspect ratio. For SCT, eight oligonucleotides are designed in such a way that adjacent strands with sticky ends can produce continuous arms in both the horizontal and vertical directions. The SCT was fabricated via free-solution annealing, and self-assembled SCT produces 2D nanolattices with periodic square cavities. All structures were observed via atomic force microscopy. Finally, we fabricated divalent nickel ion (Ni2+)- and trivalent dysprosium ion (Dy3+)-modified 2D nanolattices constructed with CT2 on a quartz substrate, and the ion coordinations were examined via Raman spectroscopy.

Roundabouts as an Effective Tool of Traffic Management

Driving speed is one of the most important factors in road safety and speed not only affects the severity of a crash, but is also related to the risk of being involved in a crash. Inappropriate speed is responsible for more than a third of all fatal accidents occurring on roads. In Poland every year majority of all traffic accidents occur in urban areas from which most is recorded at junctions and their vicinity. Hence it is of great importance to effectively manage speed and enforce speed limits on existing road network. Replacing already existing three- or four-arm junctions by roundabouts and construction new ones is considered to be a good solution for safety improvements and also are pointed to be within traffic calming features but their effectiveness is influencing by type, shape and geometry of the junction. The aim of this paper is to analyse the effectiveness of chosen traffic calming measures in comparison with the effectiveness of roundabouts. Research area was located in city of Bialystok, Poland and included a group of commonly applied physical measures together with small and mini roundabouts. Measurements of instantaneous speed by utilizing GPS data logger and assessment of the extent impact of selected TCMs on drivers’ manoeuvres were undertaken to develop the investigation. DOI: http://dx.doi.org/10.5755/j01.sace.9.4.7357

Structural and thermodynamic analysis of modified nucleosides in self-assembled DNA cross-tiles

DNA Holliday junctions are important natural strand-exchange structures that form during homologous recombination. Immobile four-arm junctions, analogs to Holliday junctions, have been designed to self-assemble into cross-tile structures by maximizing Watson–Crick base pairing and fixed crossover points. The cross-tiles, self-assembled from base pair recognition between designed single-stranded DNAs, form higher order lattice structures through cohesion of self-associating sticky ends. These cross-tiles have 16 unpaired nucleosides in the central loop at the junction of the four duplex stems. The importance of the centralized unpaired nucleosides to the structure’s thermodynamic stability and self-assembly is unknown. Cross-tile DNA nanostructures were designed and constructed from nine single-stranded DNAs with four shell strands, four arms, and a central loop containing 16 unpaired bases. The 16 unpaired bases were either 2′-deoxyribothymidines, 2′-O-methylribouridines, or abasic 1′,2′-dideoxyribonucleosides. Thermodynamic profiles and structural base-stacking contributions were assessed using UV absorption spectroscopy during thermal denaturation and circular dichroism spectroscopy, respectively, and the resulting structures were observed by atomic force microscopy. There were surprisingly significant changes in the thermodynamic and structural properties of lattice formation as a result of altering only the 16 unpaired, centralized nucleosides. The 16 unpaired 2′-O-methyluridines were stabilizing and produced uniform tubular structures. In contrast, the abasic nucleosides were destabilizing producing a mixture of structures. These results strongly indicate the importance of a small number of centrally located unpaired nucleosides within the structures. Since minor modifications lead to palpable changes in lattice formation, DNA cross-tiles present an easily manipulated structure convenient for applications in biomedical and biosensing devices.

DNA Gridiron Nanostructures Based on Four-Arm Junctions

Engineering wireframe architectures and scaffolds of increasing complexity is one of the important challenges in nanotechnology. We present a design strategy to create gridiron-like DNA structures. A series of four-arm junctions are used as vertices within a network of double-helical DNA fragments. Deliberate distortion of the junctions from their most relaxed conformations ensures that a scaffold strand can traverse through individual vertices in multiple directions. DNA gridirons were assembled, ranging from two-dimensional arrays with reconfigurability to multilayer and three-dimensional structures and curved objects.

In vivo cloning of artificial DNA nanostructures

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.

Specific Binding of Poly(ADP-ribose) Polymerase-1 to Cruciform Hairpins

Poly(ADP-ribose) polymerase-1 (PARP-1) participates in DNA cleavage and rejoining-dependent reactions, such as DNA replication, recombination and repair. PARP-1 is also important in transcriptional regulation, although the determinants for its binding to undamaged genomic DNA have not been defined. Previously, we have shown by low-resolution mapping that PARP-1 may bind to the cruciform-forming regions of its own promoter. Here, using DNase I and nuclease P(1) footprinting and atomic force microscopy, we show that PARP-1 binds to stem/loop boundaries of cruciform hairpins. Cleavage of the cruciform by the junction resolvase T4 endonuclease VII is independent of PARP-1, which indicates that PARP-1 does not bind to the four-arm junctions of the cruciform. Thus, PARP-1 differs from other cruciform-binding proteins by binding to hairpin tips rather than to junctions. Furthermore, our data indicate that PARP-1 can interact with the gene regulatory sequences by binding to the promoter-localized cruciforms.

Magnetization patterns of permalloy networks

Magnetization patterns in Permalloy network junctions are obtained by use of experiments and micromagnetic simulations. In the as-deposited thin film of the honeycomb network composed of three-arm junctions, only the lowest energy configuration, which possesses /spl plusmn/1 reduced net magnetic pole density in the junction center, is observed. In the as-deposited thin film of the square network composed of four-arm junctions, the configuration with the lowest energy density is the most frequently observed experimentally. The pattern of the independently existent antivortex, which has higher energy density, is for the first time observed experimentally in the square network.

Tensegrity: construction of rigid DNA triangles with flexible four-arm DNA junctions.

A tensegrity strategy has been explored to construct a rigid geometrical structure (triangles) from flexible DNA four-arm junctions. The resulting DNA triangles could self-assemble into 1D and 2D arrays. This tensegrity strategy is expected to play an important role in the design of biomimetic nanomaterials.

Resolution of undistorted symmetric immobile DNA junctions by vaccinia topoisomerase I.

Holliday junctions are intermediates in genetic recombination. They consist of four strands of DNA that flank a branch point. In natural systems, their sequences have 2-fold (homologous) sequence symmetry. This symmetry enables the molecules to undergo an isomerization, known as branch migration, that relocates the site of the branch point. Branch migration leads to polydispersity, which makes it difficult to characterize the physical properties of the junction and the effects of the sequence context flanking the branch point. Previous studies have reported two symmetric junctions that do not branch migrate: one that is immobilized by coupling to an asymmetric junction in a double crossover context, and a second that is based on molecules containing 5',5' and 3',3' linkages. Both are flawed by distorting the structure of the symmetric junction from its natural conformation. Here, we report an undistorted symmetric immobile junction based on the use of DNA parallelogram structures. We have used a series of these junctions to characterize the junction resolution reaction catalyzed by vaccinia virus DNA topoisomerase. The resolution reaction entails cleavage and rejoining at CCCTT/N recognition sites arrayed on opposing sides of the four-arm junction. We find that resolution is optimal when the scissile phosphodiester (Tp/N) is located two nucleotides 5' to the branch point on the helical strand. Covalent topoisomerase-DNA adducts are precursors to recombinant strands in all reactions, as expected. Kinetic analysis suggests a rate limiting step after the first-strand cleavage.

DNA-templated self-assembly of protein arrays and highly conductive nanowires.

A DNA nanostructure consisting of four four-arm junctions oriented with a square aspect ratio was designed and constructed. Programmable self-assembly of 4 x 4 tiles resulted in two distinct lattice morphologies: uniform-width nanoribbons and two-dimensional nanogrids, which both display periodic square cavities. Periodic protein arrays were achieved by templated self-assembly of streptavidin onto the DNA nanogrids containing biotinylated oligonucleotides. On the basis of a two-step metallization procedure, the 4 x 4 nanoribbons acted as an excellent scaffold for the production of highly conductive, uniform-width, silver nanowires.

Assemble four-arm DNA junctions into nanoweb

DNA is of structural polymorphism, which is useful in nanoarchitecture; especially, four-arm DNA junctions can be used to assemble nanowebs. The static four-arm DNA junctions were designed and synthesized. One-arm DNA and two-arm DNA came out simultaneously with the four-arm DNA junction’s formation. A new method, termed the two-step method, was proposed and the productivity of four-arm DNA junctions was increased. A nanoweb was assembled successfully, but it showed irregularity itself. It was not the same as we expected. We consider that it is a result from the flexibility of four-arm DNA junction.

Effect of Magnesium on Cruciform Extrusion in Supercoiled DNA

Recently, it was reported that Mg2+greatly facilitates cruciform extrusion in the short palindromes of supercoiled DNA, thereby allowing the formation of cruciform structures in vivo. Because of the potential biological importance of this phenomenon, we undertook a broader study of the effect of Mg2+on a cruciform extrusion in supercoiled DNA. The method of two-dimensional gel electrophoresis was used to detect the cruciform extrusion both in the absence and in the presence of these ions. Our results show that Mg2+shifts the cruciform extrusion in the d(CCC(AT)16GGG) palindrome to a higher, rather than to a lower level of supercoiling. In order to study possible sequence-specific properties of the short palindromes for which the unusual cruciform extrusion in the presence Mg2+was reported, we constructed a plasmid with a longer palindromic region. This region bears the same sequences in the hairpin loops and four-arm junction as the short palindrome, except that the short stems of the hairpins are extended. The extension allowed us to overcome the limitation of our experimental approach which cannot be used for very short palindromes. Our results show that Mg2+also shifts the cruciform extrusion in this palindrome to a higher level of supercoiling. These data suggest that cruciform extrusion in the short palindromes at low supercoiling is highly improbable. We performed a thermodynamic analysis of the effect of Mg2+on cruciform extrusion. The treatment accounted for the effect of Mg2+on both free energy of supercoiling and the free energy of cruciform structure per se. Our analysis showed that although the level of supercoiling required for the cruciform extrusion is not reduced by Mg2+, the ions reduce the free energy of the cruciform structure.

Triple-helical DNA as a reversible block of the branch point in a partially symmetrical DNA four-arm junction

DNA branch migration is a fundamental process in genetic recombination. A new model system has been developed for studying branch migration in a small synthetic four-arm junction. A mathematical method for describing branch-point movement by discrete steps in such junctions is also presented. The key to our experimental system is the ability to fix the location of the branch point during the assembly of the junction with a reversible block. The block is provided by a short oligonucleotide that forms triplex DNA adjacent to the initial location branch point at low pH. Raising the pH causes the triplex strand to dissociate, making the branch point free to migrate. Once mobile, the branch point can run off the end of the junction. The time-course for this runoff is consistent with a random walk of the branch point. If it is assumed that one migration step moves the branch point one base-pair, the time-course gives a rate constant for one step of 1.4 second−1 at 37°C in 10 mM MgCl2, 50 mM NaCl. These values are consistent with other measurements of non-enzymatic branch migration. We have also monitored the spread of the branch points directly with T4 endonuclease VII. Using EcoRI restriction endonuclease, we have shown that the binding of this protein to the arms of the junction essentially blocks branch migration through the binding site. In these experiments Ca2+ replaces Mg2+, and the enzyme does not cleave the DNA. In vivo there must be a special process to get branch points to migrate past bound proteins.