Branched DNA Molecules Research Articles

Two Dimensions and Two States in DNA Nanotechnology

The construction of periodic matter and nanomechanical devices are central goals of DNA nanotechnology. The minimal requirements for components of designed crystals are [1] programmable interactions, [2] predictable local intermolecular structures and [3] rigidity. The sticky-ended association of DNA molecules fulfills the first two criteria, because it is specific and diverse, and it results in the formation of B-DNA. Stable branched DNA molecules permit the formation of networks, but individual single branches are too flexible. Antiparallel DNA double crossover (DX) molecules can provide the necessary rigidity, so we use these components to tile the plane. It is possible to include DNA hairpins that act as topographic labels for this 2-D crystalline array, because they protrude from its plane. By altering sticky ends, it is possible to change the topographic features formed by these hairpins, and to detect these changes by means of AFM. We can modify arrays by restricting hairpins or by adding them to sticking ends protruding from the array. Although individual branched junctions are unsuitable for use as crystalline components, parallelograms of four 4-arm junction molecules are sufficiently rigid that they can be used to produce 2D arrays. The arrays contain cavities whose dimensions are readily tuned by changing the edges of their parallelogram components. We have used these arrays to measure directly the angle between the helices of the Holliday junction. The rigidity of the DX motif can also be exploited to produce a nanomechanical device predicated on the B-Z transition. Two DNA double crossover molecules have been joined by a segment of DNAcapable of undergoing the B-Z transition. In the B-conformation, the unconnected helices of the two molecules are on the same side of the connecting helix, whereas in the Z conformation they are on opposite sides, leading to movements of as much as 60Å. This effect is shown by fluorescence resonance energy transfer, because dyes attached to the unconnected helices have different separations in the two states.

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.

Structure-specific binding recognition of a methanogen chromosomal protein.

The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA-binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four-way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA double helix.

Branched DNA for Quantification of Viral LOAD

This is a summary of a presentation made at the 13th International Convocation on Immunology. Nucleic acids in patient samples can be quantified directly using a solid phase nucleic acid hybridization assay based on branched DNA (bDNA) signal amplification technology. For example, HIV RNA is detected in a plasma sample by hybridization of multiple specific synthetic oligonucleotides to the target, 10 of which capture the target onto the surface of a microwell plate and 39 of which mediate hybridization of branched DNA molecules to the pol region of each HTV RNA molecule. Alkaline phosphatase-labeled probes bind to each arm of the branched DNA molecules. Detection is achieved by incubating the complex with a chemiluminescent substrate and measuring the light emission. The signal is directly proportional to the level of target nucleic acid, and the quantity of HIV RNA in a sample is determined by comparison with a 4-point standard curve. In order to ensure that different subtypes of HIV-1 were detected and quantified equally, in vitro RNA transcripts of the pol region of HIV subtypes A-F were purified and quantified by OD 260, phosphate analysis, and hyperchromicity. These characterized transcripts were then quantified using the bDNA assay. Comparisons were made using a ratio of signal per attomole for each transcript. Genetic subtypes A-F quantified within a factor of 1.5, indicating that the bDNA assay can be used to measure viral load in clinical samples regardless of genotype. Accuracy is important because several studies indicate that there may be a threshold level of virus which predicts progression of HIV disease. Detection of change in viral load is important in determining the efficacy of therapy. The bDNA assay for HCV RNA can be used to determine level of virus in HCV-infected individuals and assist in establishing prognosis prior to initiation of alpha-interferon therapy. Patients with lower levels of virus are more likely to have a sustained response to therapy. Patients who respond to treatment typically have a rapid decline in virus load within one to four weeks of the start of therapy. Many patients relapse when therapy is discontinued as evidenced by a rise in virus load to near pre-treatment levels. Sustained response is most often seen with patients who have lower pre- treatment levels of RNA.

The design and engineering of nucleic acid nanoscale assemblies

It is possible to design DNA molecules that can form unusual structures and topologies. Stable DNA-branched junctions have been used to construct polyhedral catenated molecules with the connectivities of a cube and of a truncated octahedron. The truncated octahedron has been constructed following a solid-support-based methodology. Branched-DNA molecules are flexible, suggesting that triangular and deltahedral DNA objects should be favored as the components of two- and three-dimensional nucleic acid arrays. DNA polyhedra are complex catenanes. The engineering of single-stranded DNA knots and catenanes exploits the fact that a node can be equated with a half-turn of DNA.

Identification of double holliday junctions as intermediates in meiotic recombination

During meiosis, branched DNA molecules containing information from both parental chromosomes occur in vivo at loci where meiosis-specific double-stranded breaks occur. We demonstrate here that these joint molecules are recombination intermediates: they contain single strands that have undergone exchange of information. Moreover, these joint molecules are resolved into both parental and recombinant duplexes when treated in vitro with Holliday junction-resolving endonucleases RuvC or T4 endo VII. Taken together with previous observations, these results strongly suggest that joint molecules are double Holliday junctions.

Evaluation of branched DNA signal amplification for the detection of hepatitis C virus RNA.

There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 10(7)-10(8) molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%.(ABSTRACT TRUNCATED AT 400 WORDS)

Construction of branched DNA (bDNA) molecules by chemical ligation

Chemical ligation has been investigated in the assembly of branched DNA (bDNA) molecules. Water soluble coupling reagents were used to ligate single-stranded linear oligomers to bDNA molecules containing 5 and 15 branches. The ligations were particularly efficient when one of the oligomers was partially double-stranded constructed by branching to create a terminal ligation linker (AutoLink). Branched DNA molecules containing more than 1000 nucleotides were assembled.

Meiosis-specific formation of joint DNA molecules containing sequences from homologous chromosomes

All recombination models postulate one or more recombination intermediates that are joint molecules containing two homologous parental molecules. A spike of branched DNA molecules not seen in DNA from mitotic cells was found in the two-dimensional gel analysis of meiotic DNA from S. cerevisiae. The mass of molecules in the spike, the timing of its appearance and disappearance, and its absence from a recombination-defective spo11 mutant are consistent with the hypothesis that it contains recombination intermediates. The spike changes in mass as predicted for joint molecules containing DNA from homologous chromosomes rather than sister chromatids in a strain heterozygous for an RFLP. Finally, joint molecules containing DNA from homeologous chromosomes were not found, suggesting that the block to recombination between homeologous sequences occurs prior to the formation of joint molecules.

The Control of Dna Structure and Topology: An Overview

ABSTRACTThe control of structure on the nanoscale relies on intermolecular interactions whose specificity and geometry can be treated on a predictive basis. DNA fulfills this criterion, and provides an extremely convenient construction medium: The sticky-ended association of DNA molecules occurs with high specificity, and it results in the formation of double helical DNA, whose structure is well known. The use of stable branched DNA molecules permits one to make stick-figures. We have used this strategy to construct in solution a covalently closed DNA molecule whose helix axes have the connectivity of a cube: The molecule has twelve double helical edges; every edge is two helical turns in length, resulting in a hexacatenane, each of whose strands corresponds to a face of the object. We have developed a solid-support-based synthetic methodology that is more effective than solution synthesis. The key features of the technique are control over the formation of each edge of the object, and the topological closure of each intermediate. The isolation of individual objects on the surface of the support eliminates cross-reactions between growing products. The solid-support-based methodology has been used to construct a molecule whose helix axes have the connectivity of a truncated octahedron. This figure has 14 faces, of which six are square and eight are hexagonal; this Archimedean polyhedron contains 24 vertices and 36 edges, and is built from a 14-catenane of DNA. Knotted molecules appear to be the route for cloning DNA objects. It is possible to construct three knotted topologies, as well as a simple cyclic molecule from a single precursor, by control of solution conditions. Control of both branching and braiding topology is strong in this system, but control of 3-D structure remains elusive. Our key aim is the formation of prespecified 2-D and 3-D periodic structures for use in diffraction experiments. Another application envisioned is scaffolding for the assembly of molecular electronic devices.

Function of Gene 49 of Bacteriophage T4 III, Isolation of Holliday Structures from Very Fast-Sedimenting DNA

Branched DNA molecules were identified in ClaI digests of cytosine containing very fast-sedimenting DNA (VFSDNA CYT) which was isolated from Escherichia coli infected with the multiple mutant 49 -GT7 of phage T4. In about 10% of randomly picked ClaI fragments branches with three arms (Y-structures) as well as four arms (Holliday structures) were seen in the electron microscope. Branched structures were absent from ClaI digests after treatment with purified endonuclease VII (gp49) in vitro.

Symmetric immobile DNA branched junctions.

Branch migration is an isomerization of Holliday recombination intermediates that arises from their homologous (2-fold) sequence symmetry. This isomerization relocates the branch point in an apparently random fashion and thereby complicates the study of the physical and structural properties of these structures. For the past decade, these properties have been studied in low-symmetry immobile junctions, whose sequence asymmetry eliminates branch migration. The asymmetric findings of many of these studies suggest the need for a system combining both immobility and symmetry. Double-crossover DNA molecules have been used to create molecules with both these properties. Immobility is achieved by flanking one crossover with a symmetric junction and the other crossover with an asymmetric junction. Close torsional coupling between the two junctions renders the symmetric junction immobile. These molecules will enable the characterization of thermodynamic, structural, dynamic, liganding, and substrate properties of symmetric branched DNA molecules in a sequence-specific fashion.

The structure of branched DNA species

Branched DNA molecules provide a challenging set of structural problems. Operationally we define branched DNA species as molecules in which double helical segments are interrupted by abrupt discontinuities, and we draw together a number of different kinds of structure in the class, including helical junctions of different orders, and base bulges (Fig. 1).

Chemical Synthesis of Nanostructures

ABSTRACTThe control of structure on the nanoscale relies on intermolecular interactions whose specificity and geometry can be treated on a predictive basis. With this criterion in mind, DNA is an extremely favorable construction medium: The sticky-ended association of DNA molecules occurs with high specificity, and it results in the formation of double helical DNA, whose structure is well known. The use of stable branched DNA molecules permits one to make stick-figures. We have used this strategy to construct a covalently closed DNA molecule whose helix axes have the connectivity of a cube. The molecule has twelve double helical edges; each edge is two helical turns in length, resulting in a hexacatenane, each of whose strands corresponds to a face of the object. The cube has been fabricated in solution, which is inefficient. We have developed a solid-support-based synthetic methodology that is much more effective. The key features of the technique are control over the formation of each edge of the object, and the topological closure of each intermediate. Each edge results from the restriction of two hairpins, which are then ligated together. The isolation of individual objects on the surface of the support permits one to use both symmetric and asymmetric sites in the formation of edges that close polygons. We have used solid-support-based methodology to construct a molecule whose helix axes have the connectivity of a truncated octahedron. This figure has 14 faces, of which six are ideally square and eight are hexagonal; this Archimedean polyhedron contains 24 vertices and 36 edges. Control of topology is strong in this system, but control of 3-D structure remains elusive. Topological control is enhanced by the use of topological protection techniques. Our key aim is the formation of prespecified 2-D and 3-D periodic structures with defined topologies. Applications envisioned include nanomanipulators and scaffolding for molecular electronic devices.

Distance distribution in a dye-linked oligonucleotide determined by time-resolved fluorescence energy transfer

Fluorescence energy transfer is potentially a useful technique for obtaining structural and dynamic information on duplex and branched DNA molecules su

Thermodynamics of DNA branching

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is +1.1(±0.4) kcal mol −1 (at 18 °C, 10 m m-Mg 2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 °C is +27.1(±1.3) kcal mol −1, while the entropy is +89 (±30) cal K −1 mol −1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent ΔCp is also observed for the formation of one mole of junction, +0.97(±0.05) kcal deg −1mol −1.

Nanoengineering with DNA

AbstractDNA is a tractable medium for controlling the structure of matter on the nanometer scale. We have explored ligating together stable branched DNA molecules to form geometrical objects. By this means, we have assembled a 3-connected molecule whose helix axes have the connectivity of a cube. The construct is a hexacatenane, each of whose cyclic strands corresponds to a face of the object. Each of its twelve edges contains a unique recognition site for cleavage by a restriction enzyme; these sites are used to demonstrate the assembly of the object. The plectonemic structure of DNA also permits the directed synthesis of molecular knots. Recently, we have constructed trefoil knots from B-DNA and an amphichiral figure-8 knot whose helical domains contain both B-DNA and Z-DNA.We have developed a solid-support methodology for the synthesis of geometrical objects. This approach provides greater control over products and topological purity, and lends itself better to automation. Branched molecules containing 3–6 double helical arms can be formed from equimolar mixtures of their component strands, thereby enabling the construction of 3–6 connected networks. The goals of this work include the construction of periodic multiply-connected networks of DNA. The aims of these DNA constructions include using them as scaffolding to build periodic macromolecular arrays for diffraction purposes, as well as directing the assembly of molecular electronic devices. There are wellcharacterized molecular transformations of DNA that make nano-scale machines feasible to build in this molecular context. These materials are likely to be useful for understanding crystallization processes and structure-function relationships.

Site-specific Interaction of the Antitumor Antibiotic Dynemicin with Branched DNA Molecules

A specific interaction of stable branched DNA molecules with the antitumor antibiotic dynemicin is reported. Dynemicin contains an anthraquinone and an enediyne unit, and belongs to the family of enediyne antitumor agents. DNA strand scission by dynemicin appears to involve interaction of the anthraquinone core with DNA and release of a phenyl diradical from the enediyne core that can abstract hydrogen atoms from the sugar phosphate backbone of DNA. The cleavage patterns of each labeled strand in two branched tetramers of four 16-mers are compared with those of the same strands in unbranched duplex controls. Differences between the profiles corresponding to scission of branched and duplex DNA molecules can be detected in most of the strands. The strongest differences define a specific site flanking the branch in each of two branched structures. At 18 degrees C, cleavage at strand positions demarcating the site of enhanced affinity in both junctions is observed to be 70-100% more efficient than at the corresponding sequence positions in the control duplex DNA molecules. The patterns of preferential cleavage at these sites are significantly altered in the presence of excess propidium diiodide, an intercalative drug.

The use of branched DNA for nanoscale fabrication

An approach to molecular nanotechnology is presented that utilizes branched DNA molecules to form stick figures. These branched molecules are directed to associate in a particular fashion by utilizing the addressability of DNA sticky ends. The sequences of molecules are designed using an algorithm that minimizes sequence symmetry. This system has been used to construct macrocycles, a DNA quadrilateral, a molecule with the connectivity of a cube and a single-stranded DNA knot. Possible uses for this system include the construction of macromolecular zeolites for structure determination by diffraction, the assembly of new catalysts, the solubilization and delivery of drugs, and the assembly of molecular electronic devices.