DNA Toroids Research Articles

Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

Energetic preference and topological constraint effects on the formation of DNA twisted toroidal bundles.

DNA toroids are compact torus-shaped bundles formed by one or multiple DNA molecules being condensed from the solution due to various condensing agents. It has been shown that the DNA toroidal bundles are twisted. However, the global conformations of DNA inside these bundles are still not well understood. In this study, we investigate this issue by solving different models for the toroidal bundles and performing replica-exchange molecular dynamics (REMD) simulations for self-attractive stiff polymers of various chain lengths. We find that a moderate degree of twisting is energetically favorable for toroidal bundles, yielding optimal configurations of lower energies than for other bundles corresponding to spool-like and constant radius of curvature arrangements. The REMD simulations show that the ground states of the stiff polymers are twisted toroidal bundles with the average twist degrees close to those predicted by the theoretical model. Constant-temperature simulations show that twisted toroidal bundles can be formed through successive processes of nucleation, growth, quick tightening, and slow tightening of the toroid, with the two last processes facilitating the polymer threading through the toroid's hole. A relatively long chain of 512 beads has an increased dynamical difficulty to access the twisted bundle states due to the polymer's topological constraint. Interestingly, we also observed significantly twisted toroidal bundles with a sharp U-shaped region in the polymer conformation. It is suggested that this U-shaped region makes the formation of twisted bundles easier by effectively reducing the polymer length. This effect can be equivalent to having multiple chains in the toroid.

Measuring DNA extension during unfolding in a centrifugal force microscope

DNA in sperm cells can form compact toroids when bound with proteins from the protamine family. To study DNA folding and toroid formation, we have built a centrifugal force microscope (CFM) to unfold DNA and observe the intermediate folding states. In a CFM, micron-sized beads tethered to the sample by a DNA strand are placed into a microscope within a desktop centrifuge. The centrifuge applies a centrifugal force (a pseudoforce caused by inertia) to our sample, unfolding the DNA. Recording and analyzing the bead motion can yield information on the extension of the DNA over time and the intermediate stages of DNA condensation. Our CFM is operational at low centrifuge speeds, but we did not have a method for measuring the extension of the DNA. Here, we will discuss our progress in creating a program that gathers and analyzes video microscopy data from the CFM to measure extension in real time. This program will be useful in collecting data on the pathway of DNA condensation and for other force spectroscopy measurements with the CFM.

Indications of a more complex DNA toroid formation.

The compaction of DNA in sperm is at semi-crystalline levels, reaching nearly the physical condensation limit. This incredible compaction is carried out by a positively charged condensing agent, the protein protamine. Many protamines bind to the negatively charged DNA backbone, each creating a bend that eventually forms a loop. Our goal is to determine how protamine compacts the DNA even further to form toroids. Using atomic force microscopy (AFM), we imaged samples of varying lengths of DNA (1-48 kbp) under varying concentrations of protamine (0.1-3 μM). This allowed us to isolate folding states in the toroid formation pathway. It is thought that toroids are formed loop by loop and create a hexagonally packed toroid. However, we observed structures with many unstacked loops that look like flowers, vertical stacks of several loops, rod-like structures with approximately the thickness of a toroid, and toroids with nonuniform diameter and height. These structures suggest that the DNA toroid formation pathway could be more complex than we first thought. Could there be multiple pathways for the formation of toroids? Here we will present our progress toward determining toroid formation mechanisms. Understanding the physics of DNA compaction into a toroid may have interesting nanoengineering applications.

Twist-induced local curvature of filaments in DNA toroids

DNA toroidal bundles form upon condensation of one or multiple DNA filaments. DNA filaments in toroidal bundles are hexagonally packed, and collectively twist around the center line of the toroid. In a previous study, we and our coworkers argue that the filaments' curvature locally correlates with their density in the bundle, with the filaments less closely packed where their curvature appears to be higher. We base our claim on the assumption that twist has a negligible effect on the local curvature of filaments in DNA toroids. However, this remains to be proven. We fill this gap here, by calculating the distribution of filaments' curvature in a geometric model of twisted toroidal bundle, which we use to describe DNA toroids by an appropriate choice of parameters. This allows us to substantiate our previous study and suggest directions for future experiments.

Local structure of DNA toroids reveals curvature-dependent intermolecular forces.

In viruses and cells, DNA is closely packed and tightly curved thanks to polyvalent cations inducing an effective attraction between its negatively charged filaments. Our understanding of this effective attraction remains very incomplete, partly because experimental data is limited to bulk measurements on large samples of mostly uncurved DNA helices. Here we use cryo electron microscopy to shed light on the interaction between highly curved helices. We find that the spacing between DNA helices in spermine-induced DNA toroidal condensates depends on their location within the torus, consistent with a mathematical model based on the competition between electrostatic interactions and the bending rigidity of DNA. We use our model to infer the characteristics of the interaction potential, and find that its equilibrium spacing strongly depends on the curvature of the filaments. In addition, the interaction is much softer than previously reported in bulk samples using different salt conditions. Beyond viruses and cells, our characterization of the interactions governing DNA-based dense structures could help develop robust designs in DNA nanotechnologies.

DNA Toroids form via a Flower Intermediate

DNA in sperm cells must undergo an extreme compaction to almost crystalline packing levels. To produce this dense packing, DNA is condensed by protamine, a positively charged protein that loops the DNA into a toroid. Our goal is to determine the pathway and mechanism for toroid formation. We first imaged short-length (L=217-1023 nm) DNA molecules in 0-5.0 $\mu$M protamine using an atomic force microscope (AFM). At low protamine concentrations (0.2-0.6 $\mu$M), molecules dramatically condensed, folding into a flower structure. Dynamic folding measurements of the DNA using a tethered particle motion (TPM) assay revealed a corresponding, initial folding event, which was >3 loops at L=398 nm. The initial folding event was made up of smaller (<1 loop) events that had similar dynamics as protamine-induced bending. This suggests that flowers form in an initial step as protamine binds and bends the DNA. It was not until higher protamine concentrations (>2 $\mu$M) that DNA in the AFM assay formed small (<10 loop), vertically packed toroids. Taken together, these results lead us to propose a nucleation-growth model of toroid formation that includes a flower intermediate. This pathway is important in both in vivo DNA condensation and in vitro engineering of DNA nanostructures.

Mechanism of HIV-1 NC Protein-Induced Condensation of Double Stranded DNA as a Model for DNA Compaction during Reverse Transcription

Genomic RNA is packaged into the HIV-1 virion and reverse transcribed within the mature capsid into double stranded proviral DNA (vDNA) in the presence of ∼10 millimolar HIV-1 nucleocapsid protein (NC). NC is a small, highly basic protein known to condense all nucleic acids upon saturated binding. NC-induced condensation of vDNA is critical for maintaining viral capsid integrity during reverse transcription, ensuring its completion. However, how NC condenses double stranded DNA (dsDNA) is unknown. Here, we use optical tweezers to stretch single dsDNA molecules in the presence of wild-type HIV-1 NC and observe NC-induced dsDNA condensation. Above a critical concentration of NC, reducing the extension of the stretched dsDNA molecule at a constant rate reveals stepwise changes in extension along a constant-force plateau. The quantized nature of these steps suggests winding of dsDNA into a compact toroidal globule via stepwise accumulation of dsDNA in loops. We observe changes in the condensing force and kinetics of condensation and decondensation by varying NC concentration and salts, mimicking the process that occurs during capsid uncoating and gradual NC loss. Combining the results of these stretching experiments with fluorescence imaging allows us to visualize the NC-induced formation of DNA toroids in real time. These results help us understand the state of vDNA both within the capsid and after capsid uncoating.

Presence of negative supercoiling in aggregates of histone H1-plasmidic polynucleosome complexes

It was shown in the past that in the presence of histone H1, plasmidic polynucleosomes formed densely packed aggregates. Our current studies demonstrate that these aggregates are susceptible to the actions of E. coli topoisomerase I, human topoisomerase I and DNA nicking enzyme, which is the indication that negative supercoiling is present in the condensed DNA-protein complexes. Since negative supercoiling leads to formation of highly curved and compact plectonemic and toroidal DNA structures, it would be reasonable to assume that DNA negative supercoils are responsible for aggregation of histone H1-plasmidic polynucleosome complexes.

Catch-bond behavior of DNA condensate under tension**Project supported by the National Natural Science Foundation of China (Grant Nos. 11104341, 11474346, 11274374, and 61275192), the National Key Basic Research Program of China (Grant No. 2013CB837200), and the Mechanobiology Institute at National University of Singapore.

Toroid formation is an important mechanism underlying DNA condensation, which has been investigated extensively by single-molecule experiments in vitro. Here, the de-condensation dynamics of DNA condensates were studied using magnetic tweezers combined with Brownian dynamics simulations. The experimental results revealed a surprising non-monotonic dependence of the unfolding rate on the force applied under strong adhesion conditions, resembling the catch-bond behavior reported in the field of ligand-receptor interactions. Simulation results showed that the different unfolding pathways of DNA condensate under large forces derive from the force-dependent deformation of the DNA toroid, which explains the catch-bond behavior of DNA condensate in the magnetic tweezers experiments. These results challenge the universality of the regular toroidal DNA unwrapping mechanism and provide the most complete description to date of multivalent cation-dependent DNA unwrapping under tension.

Complex kinetics of DNA condensation revealed through DNA twist tracing.

Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

A simple sperm DNA toroid integrity test and risk of miscarriage.

Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N = 35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6 ± 1.7%, mean ± SEM) when compared with the miscarriage (51.2 ± 6.6%) or nonpregnant groups (60.9 ± 4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R = 0.32, P = 0.04) and pregnancy outcome (pregnant cases 73.6 ± 1.7% versus nonpregnant 58.0 ± 3.9%, P = 0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment.

DNA binding and condensation properties of the herpes simplex virus type 1 triplex protein VP19C.

Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5) interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA) and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.

Sophisticated Modeling Uncovers Atomic DNA Structure in Bacteriophage Φ29 Cavity

Sophisticated Modeling Uncovers Atomic DNA Structure in Bacteriophage Φ29 Cavity

Structural Ensemble and Dynamics of Toroidal-like DNA Shapes in Bacteriophage ϕ29 Exit Cavity

In the bacteriophage ϕ29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of ϕ29 revealed a compact toroidal DNA structure (30–40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.

Topological colloids

Smoke, fog, jelly, paints, milk and shaving cream are common everyday examples of colloids, a type of soft matter consisting of tiny particles dispersed in chemically distinct host media. Being abundant in nature, colloids also find increasingly important applications in science and technology, ranging from direct probing of kinetics in crystals and glasses to fabrication of third-generation quantum-dot solar cells. Because naturally occurring colloids have a shape that is typically determined by minimization of interfacial tension (for example, during phase separation) or faceted crystal growth, their surfaces tend to have minimum-area spherical or topologically equivalent shapes such as prisms and irregular grains (all continuously deformable--homeomorphic--to spheres). Although toroidal DNA condensates and vesicles with different numbers of handles can exist and soft matter defects can be shaped as rings and knots, the role of particle topology in colloidal systems remains unexplored. Here we fabricate and study colloidal particles with different numbers of handles and genus g ranging from 1 to 5. When introduced into a nematic liquid crystal--a fluid made of rod-like molecules that spontaneously align along the so-called 'director'--these particles induce three-dimensional director fields and topological defects dictated by colloidal topology. Whereas electric fields, photothermal melting and laser tweezing cause transformations between configurations of particle-induced structures, three-dimensional nonlinear optical imaging reveals that topological charge is conserved and that the total charge of particle-induced defects always obeys predictions of the Gauss-Bonnet and Poincaré-Hopf index theorems. This allows us to establish and experimentally test the procedure for assignment and summation of topological charges in three-dimensional director fields. Our findings lay the groundwork for new applications of colloids and liquid crystals that range from topological memory devices, through new types of self-assembly, to the experimental study of low-dimensional topology.

The Structure and Function of Highly Bent Toroidal DNA in Bacteriophage φ29

Tailed bacteriophages are highly efficient machines for infecting a host cell. A single virion must attach to the host surface, penetrate the cell wall, then release its double-stranded DNA genome. In these remarkable DNA delivery machines, controlling how and when the genome is released is a task of paramount importance. Recently, a three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of mature bacteriophage φ29 revealed an intriguing toroidal DNA structure contained in a small cavity below the viral capsid. This highly bent toroidal DNA supercoil is thought to be 30-40 basepairs of dsDNA and its function remains unknown.In this study, we employ an elastic rod model to simulate highly strained DNA as it is compressed within the protein cavity. The model provides estimates of force and energy required to form the toroid as well as its equilibrium conformation. Results reveal that a toroid can indeed form under the biologically relevant forces for the viral packing motor (up to 100 pN). To understand the effects of the highly stressed toroid on DNA structure, we then employ molecular dynamics (MD) simulations starting with the rod model-computed equilibrium as the initial condition. Following equilibration in MD, we construct an approximate density map using the final predicted toroid to compare with the recently published cryo-EM data. The computed density map correctly predicts the major dimensions of the toroid as well as regions of high and low density. Finally, we simulate the start of the ejection process by integrating the rod model forward in time upon sudden removal of the protein “tail knob.” The resulting fast dynamic collapse of the toroid suggests that its function could be to prime the ejection of the remaining viral genome.

Photoinduced Silver Nanoparticles/Nanorings on Plasmid DNA Scaffolds

Biological scaffolds are being actively explored for the synthesis of nanomaterials with novel structures and unexpected properties. Toroidal plasmid DNA separated from the Bacillus host is applied as a sacrificial mold for the synthesis of silver nanoparticles and nanorings. The photoirradiation method is applied to reduce Ag(I) on the plasmid. The nanoparticles are obtained by varying the concentration of the Ag(I) ion solution and the exposure time of the plasmid-Ag(I) complex under UV light at 254 nm and room temperature. It is found that the plasmid serves not only as a template but also as a reductant to drive the silver nucleation and deposition. The resulting nanoparticles have a face-centered cubic (fcc) crystal structure and 20-30 nm average diameter. The detailed mechanism is discussed, and other metals or alloys could also be synthesized with this method.

Protein-DNA Interactions Determine the Shapes of DNA Toroids Condensed in Virus Capsids

DNA toroids that form inside the bacteriophage capsid present different shapes according to whether they are formed by the addition of spermine or polyethylene glycol to the bathing solution. Spermine-DNA toroids present a convex, faceted section with no or minor distortions of the DNA interstrand spacing with respect to those observed in the bulk, whereas polyethylene glycol-induced toroids are flattened to the capsid inner surface and show a crescent-like, nonconvex shape. By modeling the energetics of the DNA toroid using a free-energy functional composed of energy contributions related to the elasticity of the wound DNA, exposed surface DNA energy, and adhesion between the DNA and the capsid, we established that the crescent shape of the toroidal DNA section comes from attractive interactions between DNA and the capsid. Such attractive interactions seem to be specific to the PEG condensation process and are not observed in the case of spermine-induced DNA condensation.

Investigating a Novel Toroid-Shaped DNA Structure Found in Mature Bacteriophage φ29

While the typical viral genome is several kilobases long, it is packed to near crystalline density within a viral capsid only tens of nanometers in diameter. In the case of bacteriophage φ29, this enormous compaction results from strong molecular motors that generate forces of approximately 100 pN. Recently, a three-dimensional cryo-electron microscopy reconstruction of mature φ29 was published. An intriguing feature in the reconstruction is a 60Å diameter toroidal DNA supercoil, estimated to be only 30-40 base pairs in length, within the cavity formed by the connector and the lower collar. The function of this highly-bent DNA, remains unknown. In this study, we use an elastic rod model to simulate the DNA inside the cavity. We learn that as more DNA is pushed into the capsid, compressive forces build until a critical load is reached and the DNA ‘buckles’ to fill the cavity thereby forming the toroidal supercoil observed in the reconstruction. We estimate the energy, forces, and torques required to form this toroidal DNA inside the cavity. Based on these results, we propose possible biological functions of this intriguing structure.