DNA Toroids Research Articles

Structure of DNA toroids and electrostatic attraction of DNA duplexes

DNA–DNA electrostatic attraction is considered as the driving force for the formation ofDNA toroids in the presence of DNA condensing cations. This attraction comes from theDNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter isin agreement with recent cryo-electron microscopy studies on DNA condensed with cobalthexammine. We treat the DNA–DNA interactions within the modern theory ofelectrostatic interaction between helical macromolecules. The size and thickness of thetoroids is calculated within a simple model; other models of stability of DNA toroids arediscussed and compared.

Toroid formation in charge neutralized flexible or semi‐flexible biopolymers: potential pathway for assembly of DNA carriers

The theoretical state diagram for semi-flexible macromolecules such as DNA predicts that a tightly wound toroid can be a stable structure. Experimentally, toroids roughly 100 nm in diameter are routinely observed for DNA in the presence of multivalent cations at low DNA concentration. Theory also predicts toroids can form between non-DNA semi-flexible polymers and multivalent counterions. This phenomenon provides a means to co-package DNA with functionalized anionic polymers to create gene delivery systems. We show using electron microscopy that non-DNA polymers (polylysine, polyglutamic acid, and dextran sulfate) form toroids when mixed with multi- or polyvalent ions of opposite charge. The non-DNA toroids are similar in diameter to ones made with DNA. The results using dextran sulfate, a semi-flexible polymer, are explained by current theory. However, theory predicts that high flexibility in polypeptides should discourage their incorporation into stable toroids. To explain these latter observations we propose that charge neutralization facilitates secondary structure formation, which confers stiffness, thereby allowing stable toroids for the polypeptides studied. We measured the secondary structure of the toroid-forming polypeptides using circular dichroism (CD). The CD spectrum indicates the polypeptides undergo transitions from non-ordered structures (random coil) to ordered secondary structures (either alpha-helix or beta-sheet) upon charge neutralization which supports the hypothesis. The type of secondary structure is dependent on the type of multivalent counterion used to form the toroids. Formation of the polypeptide toroids confers resistance to heat denaturation of the resulting polypeptide secondary structure. The CD spectrum of DNA in a toroid also is changed from that of uncomplexed DNA, but all of the counterions used to form DNA toroids created structures with similar CD spectra in the DNA region (250-290 nm). The toroid structure obtained using DNA is observed in other semi-flexible non-DNA polymers such as dextran sulfate, and also in flexible polymers such as polylysine and polyglutamic acid upon charge neutralization with multivalent counterions. In the flexible polymers we propose that this phenomenon is due to induction of secondary structure upon charge neutralization, which decreases polymer flexibility, i.e. increases polymer stiffness, to enable toroid formation. These results have significant implications for the co-assembly of non-DNA anionic polymers with DNA to create nanoscopic gene carriers.

DNA toroids: framework for DNA repair in Deinococcus radiodurans and in germinating bacterial spores.

Bacteria belonging to the family Deinococcaceae survive exposure to >1.5 megarads of ionizing irradiation or to extreme desiccation without lethality or mutagenesis (2, 31, 35). This tolerance derives from the ability of these species to accurately mend numerous double-strand DNA breaks (DSBs), thus reassembling an intact genome from hundreds of fragments in a manner that restores chromosomal continuity. The only known mechanism that enables accurate repair of DSBs in bacteria is RecA-dependent homologous recombination, whereby information lost at a lesion is restored by a homologous DNA sequence that acts as a template (22-24). As such, DNA repair via homologous recombination strictly depends upon the ability of cellular systems to perform a rapid and efficient genomewide search for homologous DNA sites (27). However, following extensive DNA fragmentation, no intact template remains. Homologous search conducted under such circumstances would necessarily entail repetitive reinspection of multiple randomly dispersed DNA fragments, rendering the process inherently futile (9a, 36). Indeed, the first phase of DNA repair in Deinococcus radiodurans was shown to be RecA independent (9), implying that this phase does not rely on homologous recombination. The high resistance of bacterial spores to irradiation and desiccation indicates that DSBs inflicted by these assaults on dormant spores are efficiently and accurately mended upon germination. However, DNA repair involving homologous search processes cannot occur in germinating spores, because bacterial spores regularly carry only one copy of their genomes (5). Consequently, germinating spores lack the template required for accurate homologous-recombination-mediated repair of DSBs.

Evidence that both kinetic and thermodynamic factors govern DNA toroid dimensions: effects of magnesium(II) on DNA condensation by hexammine cobalt(III).

Millimolar concentrations of divalent cations are shown to affect the size of toroids formed when DNA is condensed by multivalent cations. The origins of this effect were explored by varying the order in which MgCl(2) was added to a series of DNA condensation reactions with hexammine cobalt chloride. The interplay between Mg(II), temperature, and absolute cation concentration on DNA condensation was also investigated. These studies reveal that DNA condensation is extremely sensitive to whether Mg(II) is associated with DNA prior to condensation or Mg(II) is added concurrently with hexammine cobalt(III) at the time of condensation. It was also found that, in the presence of Mg(II), temperature and dilution can have opposite effects on the degree of DNA condensation. A systematic comparison of DNA condensates observed in this study clearly illustrates that, under our low-salt conditions, toroid size is determined by the kinetics of toroid nucleation and growth. However, when Mg(II) is present during condensation, toroid size can also be limited by a thermodynamic parameter (e.g., undercharging). The path dependence of DNA condensation presented here illustrates that regardless of which particular factors limit toroid growth, toroids formed under the various conditions of this study are largely nonequilibrium structures.

Controlling the size of nanoscale toroidal DNA condensates with static curvature and ionic strength.

The process of DNA condensation into nanometer-scale particles has direct relevance to several fields, including cell biology, virology, and gene delivery for therapeutic purposes. DNA condensation has also attracted the attention of polymer physicists, as the collapse of DNA molecules from solution into well defined particles represents an exquisite example of a polymer phase transition. Here we present a quantitative study of DNA toroids formed by condensation of 3 kb DNA with hexammine cobalt (III). The presence or absence of static loops within this DNA molecule demonstrates the effect of nucleation loop size on toroid dimensions and that nucleation is principally decoupled from toroid growth. A comparison of DNA condensates formed at low ionic strength with those formed in the presence of additional salts (NaCl or MgCl2) shows that toroid thickness is a salt-dependant phenomenon. Together, these results have allowed the development of models for DNA toroid formation in which the size of the nucleation loop directly influences the diameter of the fully formed toroid, whereas solution conditions govern toroid thickness. The data presented illustrate the potential that exists for controlling DNA toroid dimensions. Furthermore, this study provides a set of data that should prove useful as a test for theoretical models of DNA condensation.

Analysis of Compacted Semiflexible Polyanions Visualized by Atomic Force Microscopy: Influence of Chain Stiffness on the Morphologies of Polyelectrolyte Complexes

The morphologies of the compacted semiflexible biological polyanions alginate, acetan, circular plasmid DNA, and xanthan were investigated using tapping mode atomic force microscopy followed by quantitative image analysis. A shape factor was calculated for each of the observed polyelectrolyte complexes and used as a basis for dividing the structures into ensembles of morphologically linear, toroidal, and globular structures for subsequent quantitative analysis. Compaction of polyanions with chitosan yielded a small fraction of the torus morphology when the persistence length, Lp, of 25 nm (acetan) was reached. For both DNA, Lp = 50 nm, and xanthan, Lp = 120 nm, it was found that the toroids make up a substantial fraction of the complexed structures formed by the given chitosan and at room temperature. Rodlike complexes were additionally observed within DNA−chitosan complexes, whereas they do not appear as a significant fraction of chitosan-complexed high-molecular-weight xanthan. The average height of the condensates was observed to be ∼2 nm for the compacted xanthan toroids, while it was determined to be ∼5 nm for compacted DNA toroids. Reducing the degree of polymerization of xanthan yielded a decrease in the fraction of toroids. Compacted xanthan at room temperature displays a number of racquets and other morphologies similar to the reported intermediate, metastable states by simulations. The reduced abundance of such structures following annealing supports the interpretation of their metastable nature.

Ringlike structure of the Deinococcus radiodurans genome: a key to radioresistance?

The bacterium Deinococcus radiodurans survives ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms. How D. radiodurans accurately reconstructs its genome from hundreds of radiation-generated fragments in the absence of an intact template is unknown. Here we show that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. We propose that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroids, radiation-generated free DNA ends are held together, which may facilitate template-independent yet error-free joining of DNA breaks.

MgCl2 Enhances Cluster Formation by Nanoscale Toroidal DNA Condensates

Multivalent cations can cause DNA to condense from its extended state in solution into high-density toroid-shaped particles. Developing methods to control the size and size distribution of DNA toroids is an important goal for the development of artificial gene delivery systems. Here we demonstrate that changes in salt conditions, prior to condensation by multivalent cations, can significantly affect DNA condensation. Specifically, millimolar concentrations of MgCl2 are shown to cause the formation of toroid clusters, whereas NaCl at the same ionic strength does not.

Cryoelectron microscopy of lambda phage DNA condensates in vitreous ice: the fine structure of DNA toroids.

DNA toroids produced by the condensation of lambda phage DNA with hexammine cobalt (III) have been investigated by cryoelectron microscopy. Image resolution obtained by this technique has allowed unprecedented views of DNA packing within toroidal condensates. Toroids oriented coplanar with the microscope image plane exhibit circular fringes with a repeat spacing of 2.4 nm. For some toroids these fringes are observed around almost the entire circumference of the toroid. However, for most toroids well-defined fringes are limited to less than one-third of the total toroid circumference. Some toroids oriented perpendicular to the image plane reveal DNA polymers organized in a hexagonal close-packed lattice; however, for other toroids alternative packing arrangements are observed. To aid interpretation of electron micrographs, three-dimensional model toroids were generated with perfect hexagonal DNA packing throughout, as well as more physically realistic models that contain crossover points between DNA loops. Simulated transmission electron microscopy images of these model toroids in different orientations faithfully reproduce most features observed in cryoelectron micrographs of actual toroids.

Poly(amidoamine)s as potential nonviral vectors: ability to form interpolyelectrolyte complexes and to mediate transfection in vitro.

Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity. PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape. The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro. PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10,500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn approximately 8000) were studied. All PAAs retarded the electrophoretic mobility of lambda Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM. DNase II inhibition was observed. At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 +/- 6.1%), ISA 4 (92.2 +/- 11.2%), ISA 22 (69.4 +/- 3.7%), and ISA 23 (58.0 +/- 10.0%). PAAs demonstrated the ability to mediate pSV beta-galactosidase transfection of HepG2 cells. At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE. These properties suggest that PAAs warrant further development as endosomolytic vectors.

Lipoic acid-derived amphiphiles for redox-controlled DNA delivery

Background: Intracellular release of free DNA from the vector complex is one of the critical steps limiting the efficiency of non-viral gene delivery. The complex should be stable enough to prevent DNA degradation but it should be destabilized inside the cell to allow DNA release and transcription. Destabilization and degradation of synthetic vectors is also required to reduce their cytotoxicity and augment the life-time of transfected cells. Results: Here we describe new cationic amphiphiles made from the natural pro-vitamin, lipoic acid, that reversibly binds and releases DNA, depending on the redox state of the lipoate moieties. In the oxidized state these amphiphiles condense DNA into homogeneous spherical particles, which, upon reduction, swell into DNA toroids with subsequent release of free DNA. Complex reduction and DNA release can be induced by various thiols as well as enzymatically, by thioredoxin reductase. Transfection with amphiphile–DNA complexes in vitro shows a several fold increase of transgene expression compared with DOTAP, and can be further augmented by attachment of the nucleus-targeting peptide to the amphiphile. The increase of transfection efficiency results from GSH- and NAD(P)H-dependent complex reduction and release of free DNA inside the cells. Conclusions: The present work demonstrates the principle of a redox-controlled gene delivery system that uses the reversibility of thiol–disulfide exchange reaction. Our data suggest that the efficiency of synthetic vectors can be augmented by their controlled destabilization inside the cells. Being formed from the natural non-toxic compound lipoic acid, these cationic amphiphiles provide a new promising class of synthetic vectors for gene delivery.

Observation of DNA–polymer condensate formation in real time at a molecular level

Dynamic real time assembly of toroidal and rod-like DNA condensates has been visualised using atomic force microscopy. Imaging has been conducted in an aqueous environment allowing the visualisation of hydrated, pegylated-polymer DNA condensates undergoing dynamic structural movement and conformational change. A major hurdle in the field of gene delivery is cellular transfection and the subsequent transfer of condensed genetic material to the cell nucleus. An increased understanding of the process of DNA condensation will aid the development and optimisation of gene delivery vectors.

Nucleation of DNA Condensation by Static Loops: Formation of DNA Toroids with Reduced Dimensions

Nucleation of DNA Condensation by Static Loops: Formation of DNA Toroids with Reduced Dimensions

TOROIDAL DNA: TOPOLOGY, GEOMETRY AND ELECTROSTATICS

Toroidal DNA condensates have received considerable attention as a model of ordered DNA condensation. Geometry and topology of fibre bundles can be efficient tools in order to study these structures. Such methods have given fruitful results when considering toroidal vesicles made of amphiphile films and blue phases structures observed in liquid crystals made of long chiral molecules. We give, in this paper, the geometrical arguments supporting this description. Then, we show how electrostatic interactions could be studied using curved spaces. We show that it is possible to have conditions resulting in attractive interaction between DNA molecules in the presence of positive charges.

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.

High-Yield Preparation of Oligomeric C-Type DNA Toroids and Their Characterization by Cryoelectron Microscopy

The quantitative and reversible compaction of open circular plasmid DNA (7676 bp) into toroids containing one to 19 molecules by sequential treatment with spermine and an excess of uranyl acetate is reported. The toroidal DNA structure was proven by cryoelectron microscopy. Linearized and supercoiled variants of the DNA also gave toroids under these conditions, but yields were significantly lower. In the presence of spermine alone no toroids were found. Open circular plasmid B-DNA helix was converted into the C-type helical form upon compaction as was shown by CD spectroscopy (negative peak at 255 nm) and electron microscopy (1.8-nm interduplex distance instead of 2.9 nm). Addition of uranyl salt to the DNA−spermine complexes resulted in the formation of netlike assemblies which further compacted to give toroids. A model containing a hexagonal arrangement of DNA strands with extensive strand crossings is proposed. Curvature and thus toroid formation is thought to be induced by the hydrophobic DNA coating ...

P-B1-09 Shape and structure of toroidal DNA condensates: Ubbink J, Odijk T. Department of Chemical Engineering and Materials Science Delft University of Technology (NL)

P-B1-09 Shape and structure of toroidal DNA condensates: Ubbink J, Odijk T. Department of Chemical Engineering and Materials Science Delft University of Technology (NL)

Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model

Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent will all available data. Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure.

A constant radius of curvature model for the organization of DNA in toroidal condensates.

Toroidal DNA condensates have received considerable attention for their possible relationship to the packaging of DNA in viruses and in general as a model of ordered DNA condensation. A spool-like model has primarily been supported for DNA organization within toroids. However, our observations suggest that the actual organization may be considerably different. We present an alternate model in which DNA for a given toroid is organized within a series of equally sized contiguous loops that precess about the toroid axis. A related model for the toroid formation process is also presented. This kinetic model predicts a distribution of toroid sizes for DNA condensed from solution that is in good agreement with experimental data.

Elastic model of DNA supercoiling in the infinite-length limit

The energies of plectonemic and toroidal supercoiled DNA are calculated by treating DNA as an elastic rod with a finite radius. End effects are ignored and all extensive quantities (e.g., writhe, bend energy) are treated as linear densities (writhe per unit length, bend energy per unit length). Minimum energy configurations are found. For plectonemic DNA, the superhelical pitch angle α is in the range 45°<α≤90°. For low values of specific linking difference, most superhelicity is in writhe. As specific linking difference increases, a greater proportion of superhelicity is in twist. Under physiological conditions, roughly 88% of superhelicity is in writhe. Ionic strength effects are discussed, and it is found that variation of excluded volume with ionic strength has a large effect, resulting in significantly greater torsional stress in supercoiled DNA at low ionic strength. For biologically relevant values of specific linking difference, the plectonemic conformation is energetically favored over toroidal conformations. Results are compared with electron microscopy data. The application of the model to DNA conformational transitions is discussed.