Quantifying the impact of simple DNA parameters on the cyclization J-factor for single-basepair-addition families.

We use Monte Carlo simulation to quantify the change in cyclization J-factor within a dramatically simplified model of DNA that involves parameters for uniform stiffnesses, intrinsic twist, and intrinsic bending (including nonplanar bending). Plots of J versus DNA length over multiple periods of helical repeat are fit to a simple functional form in order to project the behavior of J over a broad range of these model parameters. In some instances, this process allows us to find families of DNA molecules (within our model) with quite different material properties, but very similar plots of J versus length, so similar as to likely to be indistinguishable by experiments. This effect is seen both for the parameter-pair of bend angle and stiffness scaling, as well as for the parameter-trio of helical repeat, bend angle, and bend non-planarity.

A Monte Carlo Study of Knots in Long Double-Stranded DNA Chains.

We determine knotting probabilities and typical sizes of knots in double-stranded DNA for chains of up to half a million base pairs with computer simulations of a coarse-grained bead-stick model: Single trefoil knots and composite knots which include at least one trefoil as a prime factor are shown to be common in DNA chains exceeding 250,000 base pairs, assuming physiologically relevant salt conditions. The analysis is motivated by the emergence of DNA nanopore sequencing technology, as knots are a potential cause of erroneous nucleotide reads in nanopore sequencing devices and may severely limit read lengths in the foreseeable future. Even though our coarse-grained model is only based on experimental knotting probabilities of short DNA strands, it reproduces the correct persistence length of DNA. This indicates that knots are not only a fine gauge for structural properties, but a promising tool for the design of polymer models.

Amplified stretch of bottlebrush-coated DNA in nanofluidic channels.

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.

Assessment of the DNA damage using the fluorescence microscope.

DNA damage was monitored by fluorescent microscopy observations of DNA fluorescent images after hydrodynamic stretching on a microscope glass. DNA double-strand breaks lead to a decrease of the average length of observed fluorescent DNA molecules. Compared to conventional methods such as electrophoresis, the proposed method allows for the analysis of the DNA damage at very low DNA breaking frequency. In particular, this method was used to study DNA damage by weak UV irradiation in solutions of quantum dots.

50 nm DNA nanoarrays generated from uniform oligonucleotide films.

One of the most challenging but potentially rewarding goals in nanoscience is the ability to direct the assembly of nanoscale materials into functional architectures with high yields, minimal steps, and inexpensive procedures. Despite their unique physical properties, the inherent difficulties of engineering wafer-level arrays of useful devices from nanoscale materials in a cost-effective manner have provided serious roadblocks toward technological impact. To address nanoscale features while still maintaining low fabrication costs, we demonstrate here an inexpensive printing method that enables repeated patterning of large-area arrays of nanoscale materials. DNA strands were patterned over 4 mm areas with 50 nm resolution by a soft-lithographic subtraction printing process, and DNA hybridization was used to direct the assembly of sub-20 nm materials to create highly ordered two-dimensional nanoparticle arrays. The entire printing and assembly process was accomplished in as few as three fabrication steps and required only a single lithographically templated silicon master that could be used repeatedly. The low-cost procedures developed to generate nanoscale DNA patterns can be easily extended toward roll-to-roll assembly of nanoscale materials with sub-50 nm resolution and fidelity.

Microscopic mechanics of hairpin DNA translocation through synthetic nanopores.

Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and--in two distinct orientations--stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.

Healing assessment of tile sets for error tolerance in DNA self-assembly.

An assessment of the effectiveness of healing for error tolerance in DNA self-assembly tile sets for algorithmic/nano-manufacturing applications is presented. Initially, the conditions for correct binding of a tile to an existing aggregate are analysed using a Markovian approach; based on this analysis, it is proved that correct aggregation (as identified with a so-called ideal tile set) is not always met for the existing tile sets for nano-manufacturing. A metric for assessing tile sets for healing by utilising punctures is proposed. Tile sets are investigated and assessed with respect to features such as error (mismatched tile) movement, punctured area and bond types. Subsequently, it is shown that the proposed metric can comprehensively assess the healing effectiveness of a puncture type for a tile set and its capability to attain error tolerance for the desired pattern. Extensive simulation results are provided.

Monte carlo simulations of protein assembly, disassembly, and linear motion on DNA.

We use Monte Carlo simulations to analyze the simultaneous interactions of multiple proteins to a long DNA molecule. We study the time dependence of protein organization on DNA for different regimes that comprise (non)cooperative sequence-independent protein assembly, dissociation, and linear motion. A range of different behaviors is observed for the dynamics, final coverage, and cluster size distributions. We observe that the DNA substrate is almost never completely covered by protein when taking into account only (non)cooperative binding, because gaps remain on the substrate that are smaller than the binding site size of the protein. Due to these gaps, the apparent binding size of a protein during noncooperative binding can be overestimated by up to 30%. During dissociation of cooperatively bound proteins, the dissociation curve can be exponentially shaped even when allowing only end-dependent dissociation. We discuss the potential of our method for the analysis of a number of single-molecule experiments, for example, the binding of the DNA-repair proteins RecA and Rad51 to DNA.

Implementing EM and Viterbi algorithms for Hidden Markov Model in linear memory.

BACKGROUND: The Baum-Welch learning procedure for Hidden Markov Models (HMMs) provides a powerful tool for tailoring HMM topologies to data for use in knowledge discovery and clustering. A linear memory procedure recently proposed by Miklós, I. and Meyer, I.M. describes a memory sparse version of the Baum-Welch algorithm with modifications to the original probabilistic table topologies to make memory use independent of sequence length (and linearly dependent on state number). The original description of the technique has some errors that we amend. We then compare the corrected implementation on a variety of data sets with conventional and checkpointing implementations. RESULTS: We provide a correct recurrence relation for the emission parameter estimate and extend it to parameter estimates of the Normal distribution. To accelerate estimation of the prior state probabilities, and decrease memory use, we reverse the originally proposed forward sweep. We describe different scaling strategies necessary in all real implementations of the algorithm to prevent underflow. In this paper we also describe our approach to a linear memory implementation of the Viterbi decoding algorithm (with linearity in the sequence length, while memory use is approximately independent of state number). We demonstrate the use of the linear memory implementation on an extended Duration Hidden Markov Model (DHMM) and on an HMM with a spike detection topology. Comparing the various implementations of the Baum-Welch procedure we find that the checkpointing algorithm produces the best overall tradeoff between memory use and speed. In cases where sequence length is very large (for Baum-Welch), or state number is very large (for Viterbi), the linear memory methods outlined may offer some utility. CONCLUSION: Our performance-optimized Java implementations of Baum-Welch algorithm are available at http://logos.cs.uno.edu/~achurban. The described method and implementations will aid sequence alignment, gene structure prediction, HMM profile training, nanopore ionic flow blockades analysis and many other domains that require efficient HMM training with EM.

Efficient chain moves for Monte Carlo simulations of a wormlike DNA model: excluded volume, supercoils, site juxtapositions, knots, and comparisons with random-flight and lattice models.

We develop two classes of Monte Carlo moves for efficient sampling of wormlike DNA chains that can have significant degrees of supercoiling, a conformational feature that is key to many aspects of biological function including replication, transcription, and recombination. One class of moves entails reversing the coordinates of a segment of the chain along one, two, or three axes of an appropriately chosen local frame of reference. These transformations may be viewed as a generalization, to the continuum, of the Madras-Orlitsky-Shepp algorithm for cubic lattices. Another class of moves, termed T+/-2, allows for interconversions between chains with different lengths by adding or subtracting two beads (monomer units) to or from the chain. Length-changing moves are generally useful for conformational sampling with a given site juxtaposition, as has been shown in previous lattice studies. Here, the continuum T+/-2 moves are designed to enhance their acceptance rate in supercoiled conformations. We apply these moves to a wormlike model in which excluded volume is accounted for by a bond-bond repulsion term. The computed autocorrelation functions for the relaxation of bond length, bond angle, writhe, and branch number indicate that the new moves lead to significantly more efficient sampling than conventional bead displacements and crankshaft rotations. A close correspondence is found in the equilibrium ensemble between the map of writhe computed for pair of chain segments and the map of site juxtapositions or self-contacts. To evaluate the more coarse-grained freely jointed chain (random-flight) and cubic lattice models that are commonly used in DNA investigations, twisting (torsional) potentials are introduced into these models. Conformational properties for a given superhelical density sigma may then be sampled by computing the writhe and using White's formula to relate the degree of twisting to writhe and sigma. Extensive comparisons of contact patterns and knot probabilities of the more coarse-grained models with the wormlike model show that the behaviors of the random-flight model are similar to that of DNA molecules in a solution environment with high ionic strengths, whereas the behaviors of the cubic lattice model with excluded volume are akin to that of DNA molecules under low ionic strengths.

High-throughput sequence alignment using Graphics Processing Units.

BACKGROUND: The recent availability of new, less expensive high-throughput DNA sequencing technologies has yielded a dramatic increase in the volume of sequence data that must be analyzed. These data are being generated for several purposes, including genotyping, genome resequencing, metagenomics, and de novo genome assembly projects. Sequence alignment programs such as MUMmer have proven essential for analysis of these data, but researchers will need ever faster, high-throughput alignment tools running on inexpensive hardware to keep up with new sequence technologies. RESULTS: This paper describes MUMmerGPU, an open-source high-throughput parallel pairwise local sequence alignment program that runs on commodity Graphics Processing Units (GPUs) in common workstations. MUMmerGPU uses the new Compute Unified Device Architecture (CUDA) from nVidia to align multiple query sequences against a single reference sequence stored as a suffix tree. By processing the queries in parallel on the highly parallel graphics card, MUMmerGPU achieves more than a 10-fold speedup over a serial CPU version of the sequence alignment kernel, and outperforms the exact alignment component of MUMmer on a high end CPU by 3.5-fold in total application time when aligning reads from recent sequencing projects using Solexa/Illumina, 454, and Sanger sequencing technologies. CONCLUSION: MUMmerGPU is a low cost, ultra-fast sequence alignment program designed to handle the increasing volume of data produced by new, high-throughput sequencing technologies. MUMmerGPU demonstrates that even memory-intensive applications can run significantly faster on the relatively low-cost GPU than on the CPU.

DNA looping kinetics analyzed using diffusive hidden Markov model.

Tethered particle experiments use light microscopy to measure the position of a micrometer-sized bead tethered to a microscope slide via an approximately micrometer-length polymer, to infer the behavior of the invisible polymer. Currently, this method is used to measure rate constants of DNA loop formation and breakdown mediated by repressor protein that binds to the DNA. We report a new technique for measuring these rates using a modified hidden Markov analysis that directly incorporates the diffusive motion of the bead, which is an inherent complication of tethered particle motion because it occurs on a timescale between the sampling frequency and the looping time. We compare looping lifetimes found with our method, which are consistent over a range of sampling frequencies, to those obtained via the traditional threshold-crossing analysis, which vary depending on how the raw data are filtered in the time domain. Our method does not involve such filtering, and so can detect short-lived looping events and sudden changes in looping behavior.

Monte Carlo simulation of chromatin stretching.

We present Monte Carlo (MC) simulations of the stretching of a single chromatin fiber. The model approximates the DNA by a flexible polymer chain with Debye-Hückel electrostatics and uses a two-angle zigzag model for the geometry of the linker DNA connecting the nucleosomes. The latter are represented by flat disks interacting via an attractive Gay-Berne potential. Our results show that the stiffness of the chromatin fiber strongly depends on the linker DNA length. Furthermore, changing the twisting angle between nucleosomes from 90 degrees to 130 degrees increases the stiffness significantly. An increase in the opening angle from 22 degrees to 34 degrees leads to softer fibers for small linker lengths. We observe that fibers containing a linker histone at each nucleosome are stiffer compared to those without the linker histone. The simulated persistence lengths and elastic moduli agree with experimental data. Finally, we show that the chromatin fiber does not behave as an isotropic elastic rod, but its rigidity depends on the direction of deformation: Chromatin is much more resistant to stretching than to bending.

Fabrication, characterization, and biological assessment of multilayered DNA-coatings for biomaterial purposes.

This study describes the fabrication of two types of multilayered coatings onto titanium by electrostatic self-assembly (ESA), using deoxyribosenucleic acid (DNA) as the anionic polyelectrolyte and poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Both coatings were characterized using UV-vis spectrophotometry, atomic force microscopy (AFM), X-ray photospectroscopy (XPS), contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and for the amount of DNA immobilized. The mutagenicity of the constituents of the coatings was assessed. Titanium substrates with or without multilayered DNA-coatings were used in cell culture experiments to study cell proliferation, viability, and morphology. Results of UV-vis spectrophotometry, AFM, and contact angle measurements clearly indicated the progressive build-up of the multilayered coatings. Furthermore, AFM and XPS data showed a more uniform build-up and morphology of [PDL/DNA]-coatings compared to [PAH/DNA]-coatings. DNA-immobilization into both coatings was linear, and approximated 3microg/cm(2) into each double-layer. The surface morphology of both types of multilayered DNA-coatings showed elevations in the nanoscale range. No mutagenic effects of DNA, PDL, or PAH were detected, and cell viability and morphology were not affected by the presence of either type of multilayered DNA-coating. Still, the results of the proliferation assay revealed an increased proliferation of primary rat dermal fibroblasts on both types of multilayered DNA-coatings compared to non-coated controls. The biocompatibility and functionalization of the coatings produced here, will be assessed in subsequent cell culture and animal-implantation studies.

Dynamics of DNA replication: an ultrastructural study.

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.

Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells.

PURPOSE: Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets. MATERIALS AND METHODS: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs). RESULTS: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target. CONCLUSION: Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.

Monte Carlo simulation of the assembly of bis-biotinylated DNA and streptavidin.

We present Monte Carlo simulations of the self-assembly of bivalent bis-biotinylated DNA molecules with the tetravalent biotin-binding protein streptavidin (STV). By fitting the STV binding probabilities for the four possible valencies, the modelling correctly reproduces the dependencies of various network parameters experimentally observed in an earlier study. The combined results from the experimental and theoretical studies suggest that the binding probability for divalent STV formation is about 50 times larger than for the formation of trivalent and about 200 times larger than for tetravalent STV. In accordance with the experimental results, the modelling also indicates that the mixture of an equimolar ratio of DNA and STV leads to a maximum in size of the oligomeric DNA-STV clusters formed. Furthermore, we found a percolation transition in which the DNA cluster size increases rapidly with increasing DNA concentration resulting in the formation of a single supercluster at elevated concentrations. This behaviour coincides with the occurrence of an immobile band previously observed in electrophoretic experiments, indicating the formation of extremely large DNA-STV aggregate networks.

The clinical value of sperm nuclear DNA assessment.

Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.

Computer simulation of the 30-nanometer chromatin fiber.

A new Monte Carlo model for the structure of chromatin is presented here. Based on our previous work on superhelical DNA and polynucleosomes, it reintegrates aspects of the "solenoid" and the "zig-zag" models. The DNA is modeled as a flexible elastic polymer chain, consisting of segments connected by elastic bending, torsional, and stretching springs. The electrostatic interaction between the DNA segments is described by the Debye-Hückel approximation. Nucleosome core particles are represented by oblate ellipsoids; their interaction potential has been parameterized by a comparison with data from liquid crystals of nucleosome solutions. DNA and chromatosomes are linked either at the surface of the chromatosome or through a rigid nucleosome stem. Equilibrium ensembles of 100-nucleosome chains at physiological ionic strength were generated by a Metropolis-Monte Carlo algorithm. For a DNA linked at the nucleosome stem and a nucleosome repeat of 200 bp, the simulated fiber diameter of 32 nm and the mass density of 6.1 nucleosomes per 11 nm fiber length are in excellent agreement with experimental values from the literature. The experimental value of the inclination of DNA and nucleosomes to the fiber axis could also be reproduced. Whereas the linker DNA connects chromatosomes on opposite sides of the fiber, the overall packing of the nucleosomes leads to a helical aspect of the structure. The persistence length of the simulated fibers is 265 nm. For more random fibers where the tilt angles between two nucleosomes are chosen according to a Gaussian distribution along the fiber, the persistence length decreases to 30 nm with increasing width of the distribution, whereas the other observable parameters such as the mass density remain unchanged. Polynucleosomes with repeat lengths of 212 bp also form fibers with the expected experimental properties. Systems with larger repeat length form fibers, but the mass density is significantly lower than the measured value. The theoretical characteristics of a fiber with a repeat length of 192 bp where DNA and nucleosomes are connected at the core particle are in agreement with the experimental values. Systems without a stem and a repeat length of 217 bp do not form fibers.

A method for radioprobing DNA structures using Auger electrons.

PURPOSE: To present a new method for radioprobing a DNA triple helix structure by Auger electrons emitted in the decay of 125I using theoretical/computational approaches. MATERIALS AND METHODS: A Monte Carlo track structure method was used to simulate the damage to a triplex resulting from Auger electrons emitted in the decay of an incorporated 125I atom in plasmid DNA. Comparison of the theoretical frequency distributions of single-strand breaks induced on the Pu and Py strands with the experimental data and a knowledge of the distances from the strand breaks to the iodine provide information on the structures otherwise difficult to obtain with X-ray crystallography. RESULTS: In comparing theoretical frequency distributions of single-strand breaks with the experimental data it is found that the results are very sensitive to the conformation of the triplex model used. It is found that the best fit to the experimental data results from using a hybrid triplex model, in which the base-step geometry is A-like, while the sugar puckers adopt the B-like C2'-endo conformation. CONCLUSIONS: The approach and technique presented here represent a valuable new addition to the methods available for DNA structure determination since they provide information on medium-range structure otherwize difficult to obtain in the absence of X-ray crystallography. It is concluded that currently accepted models for triplex structure are not optimal, and a modified structure is proposed that fits the radioprobing results better, while maintaining agreement with the fibre diffraction and NMR data. Although the method has proved to be very useful for scoring alternative trial solutions, further studies combining experimental data from multiple iodine positions with track structure modelling are required for directing structural optimization.