Dynamics Of Single DNA Molecules Research Articles

Electric field induced coil-stretch transition of DNA molecule

The dynamics of single DNA molecules in a homogeneous planar elongational electric field is investigated in the framework of the Hookean Dumbbell model. For a Gaussian, rapidly changing advecting flow the Fokker-Planck equation for time-dependent probability density function of DNA elongation is obtained. The analytical solution for probability density function of time independent problem is derived. The coil-stretch transition is studied when the elongation of the DNA molecule is very small than the maximum length. The characteristic relaxation time is calculated as a function of the Weissenberg number. It is observed that the coil-stretch transition and characteristic relaxation time of DNA molecule strongly depend on the angle of the principal axis of molecule with respect to axis of elongation.

Contraction and Tumbling Dynamics of DNA in Shear Flows under Confinement Induced by Transverse Viscoelastic Forces

The dynamics of single DNA molecules conveyed in a viscoelastic fluid flow with an opposing electrophoretic force are investigated by fluorescence microscopy. For balanced hydrodynamic and electrop...

Diffusion and Conformational Dynamics of Single DNA Molecules Crowded by Cytoskeletal Proteins

The high concentrations of proteins crowding cells greatly influence intracellular DNA dynamics. These crowders, ranging from small mobile proteins to large cytoskeletal filaments such as semiflexible actin and rigid microtubules, can hinder diffusion and induce conformational changes in DNA. While previous studies have mainly focused on the effect of small mobile crowders on DNA transport, we examine the impact of crowding by actin filaments and microtubules. Further, because actin filaments and microtubules are formed by polymerization of actin monomers and tubulin dimers, respectively, we also investigate the role that the polymerization state of each protein plays in DNA transport and in the time-varying conformational changes of single DNA molecules diffusing in in vitro networks of polymerized and monomeric actin and tubulin. We find that crowding by actin monomers slows DNA diffusion while tubulin crowding actually increases diffusion coefficients. Monomeric actin crowding DNA diffusion, more than when actin is polymerized, while crowding by tubulin dimers increases DNA diffusion more than when tubulin is polymerized (microtubules). Further, we find unexpected relationships between DNA coil size and diffusion when crowded. All crowding conditions lead to some degree of DNA compaction, but less compaction enables faster dynamics.

Fabrication of birefringent nanocylinders for single-molecule force and torque measurement

Optically anisotropic subwavelength scale dielectric particles have been shown to enable studies of the mechanical properties of bio-molecules via optical trapping and manipulation. However, techniques emphasized to date for fabrication of such particles generally suffer from limited uniformity and control over particle dimensions, or low throughput and high cost. Here, an approach for rapid, low-cost, fabrication of large quantities of birefringent quartz nanocylinders with dimensions optimized for optical torque wrench experiments is described. For a typical process, 108 or more quartz cylinders with diameters of 500 nm and heights of 800 nm, with uniformity of ±5% in each dimension, can be fabricated over ∼10 cm2 areas, for binding to a single bio-molecule, and harvested for use in optical trapping experiments. Use of these structures to measure extensional and torsional dynamics of single DNA molecules is demonstrated with measured forces and torques shown to be in very good agreement with previously reported results.

Dynamics and Conformation of Semiflexible Polymers in Strong Quasi-1D and -2D Confinement

We investigate the conformation and relaxation dynamics of single DNA molecules in strong confinement (smaller than persistence length) with coarse-grained semiflexible chain (SFC) models using overdamped Langevin dynamics simulations. DNA properties in nanochannels and nanoslits are studied in confinement with height (H) ranging from the DNA radius of gyration (Rg) to smaller than the persistence length (P). Qualitatively different dependences of chain conformation and relaxation time on H in moderate (P < H < Rg) and strong (H < P) confinement are observed for very stiff SFC in the nanochannel but not in the nanoslit. The chain relaxation time (trelax) exhibits strong power-law dependence in H < P nanochannels, verified with and without including hydrodynamic interactions (HI). The inclusion of hydrodynamic interactions affects chain relaxation dynamics even in strong confinement, indicating the intersegmental hydrodynamic interactions affect dominant segmental relaxation mechanisms of strongly confined...

Oxaliplatin and its enantiomer induce different condensation dynamics of single DNA molecules.

The interactions of DNA with oxaliplatin (Pt(R,R-DACH)) or its enantiomer (Pt(S,S-DACH)) were investigated using magnetic tweezers and atomic force microscope. In the process of DNA condensation induced by Pt-DACH, only diadducts and micro-loops are formed at low Pt-DACH concentrations, while at high Pt-DACH concentrations, besides the diadducts and micro-loops, long-range cross-links are also formed. The diadduct formation rate of Pt(R,R-DACH) is higher than that of Pt(S,S-DACH). However, the proportions of micro-loops and long-range cross-links for Pt(S,S-DACH) are higher than those for Pt(R,R-DACH). We propose a model to explain these differences between the effect of Pt(R,R-DACH) and that of Pt(S,S-DACH) on DNA condensation. The study has strong implications for the understanding of the effect of chirality on the interaction between Pt-DACH and DNA and the kinetics of DNA condensation induced by platinum complexes.

DNA molecule stretching through thermo-electrophoresis and thermal convection in a heated converging-diverging microchannel

A novel DNA molecule stretching technique is developed and tested herein. Through a heated converging-diverging microchannel, thermal convection and thermophoresis induced by regional heating are shown to significantly elongate single DNA molecules; they are visualized via a confocal laser scanning microscopy. In addition, electrophoretic stretching is also implemented to examine the hybrid effect on the conformation and dynamics of single DNA molecules. The physical properties of the DNA molecules are secured via experimental measurements.

Experimental study of the effect of disorder on DNA dynamics in post arrays during electrophoresis

We used top-down fabrication techniques to create both an ordered hexagonal array and a disordered array of 1 μm diameter cylindrical posts in a silicon dioxide microchannel with the same number of posts per unit area. The electrophoretic mobility and dispersion coefficient of λ DNA in each of the arrays were obtained as a function of the electric field using ensembles of DNA molecules in a double channel device that minimizes experimental artifacts. To deepen our understanding of the transport, we also used fluorescence microscopy to examine the dynamics of single DNA molecules as they interact with the arrays at a fixed value of the electric field. Based on the results of these two types of experiments, we conclude that the electrophoretic mobility is not dependent on the array order but that band broadening in the device is greater in the disordered array.

Nanofluidic Studies of DNA Compaction by Macromolecular Crowding and Architectural Protein

The study of single DNA molecules confined in a nanochannel is of importance from both biotechnological and biophysical points of view. We produce nanochannels in cheap PDMS based biochips. The two-dimensional cross-sectional diameter of the channels is in the range of 50 to 300 nm. We measure the extensions of single bacteriophage DNA molecules in various environmental conditions with fluorescence microscopy. In this contribution, two important and related issues will be addressed. The first issue is the control of conformation of DNA by macromolecular crowding. We have investigated dextran and the like-charge proteins bovine serum albumin (BSA) and hemoglobin (Hb). As a surprising result, we found that the DNA molecules take a more extended rather than a more compacted conformation in the presence of low volume fractions of dextran. At higher volume fractions, the DNA molecules collapse into a condensed state. DNA collapse was also observed for the like-charge proteins, albeit at much lower volume fractions as compared to dextran. The second issue is the effect of architectural protein. For this purpose, we have investigated the effects of the bacterial nucleoid associated H-NS and HU proteins. Our results show that the interplay of DNA/ligand interaction, osmotic pressure, like charge attraction, and anisotropic confinement is of paramount importance in controlling the conformation and compaction of DNA. The biochip provides a platform to investigate single biomolecules and is complementary to other single molecule techniques such as those based on molecular tweezers. Some other exemplary experiments on the structure and dynamics of single DNA molecules inside nanochannels, which are in progress in our laboratories, will be presented.

Equilibrium dynamics of single DNA molecules confined to nanopit structures

&#x0D; &#x0D; &#x0D; Introduction: of great interest in the physical sciences today, is the study of single molecules in nano-fluidic devices. These ‘labs-on-a-chip’ can provide the basic framework for quantifying the behavior of molecules, such as polymers, under confinement. This study is an investigation of a theoretical free-energy model used to predict thermodynamic properties of DNA molecules situated in a device called a nanopit array. Two parameters in the model, molecule length and nanopit width, are varied and tested against experimental data. Methods: Video-fluorescence microscopy was used to image single DNA molecules in the nanopit array; analysis consisted of determining the average number of nanopits occupied by a single DNA molecule over time. results: good qualitative agreement was reached between theory and experiment for the nanopit width variation, but molecule-length variation predictions were shown to still need improvement. A least-squares fit of the theory to the data suggested that the entropic parameter, A, and the excluded volume term, b, have a modified dependence on nanoslit height and nanopit depth than what is currently predicted by the model. discussion: These experiments confirm that the theoretical model is adequate under certain regimes and predicts conditions under which theory and experiment may significantly diverge. Modifications to the theory are proposed.&#x0D; &#x0D; &#x0D;

Hybridised functional micro- and nanostructure for studying the kinetics of a single biomolecule

In this Letter, the nanoporous anodic alumina membranes (nanoPAAM) were first fabricated by the two-step anodic oxidisation method, and then based on these nanoPAAM master molds, polymer polymethylmethacrylate nanopillars with controllable heights and diameters were achieved by the direct casting method. Subsequently, combined with the ultraviolet lithography or electron beam lithography, a kind of useful platform of microfluidic channel that was embedded with nanopillar arrays was achieved. Finally, the transferring processes of DNA molecules around the entrance of the hybrid channel were monitored with an epi-fluorescence microscopy and electron multiplying charge coupled device camera in real time. The hybrid structure of microchannel with nanopillar arrays provides critical advantages when using the fluorescent microscopy to detect the dynamics of single DNA molecules.

Revisiting the Conformation and Dynamics of DNA in Slitlike Confinement

We experimentally investigated the equilibrium conformation and dynamics of single DNA molecules in slitlike nanochannels. We measured the in-plane radius of gyration (R ) diffusivity (D), and longest relaxation time (τ)ofλ-DNA (48.5 kbp) as functions of the slit height using fluorescence microscopy. Our results show that the in-plane radius of gyration increases monotonically with decreasing slit height, in contrast to results from Bonthuis et al. 15 but in agreement with our simulations and those of other groups. In strongconfinement (slit height <100nm), thescaling ofD, τ, andR )with slitheight does notshow anevident change, suggesting that the transition from the de Gennes regime to the Odijk regime is gradual and broad.

Single-molecule DNA dynamics in tapered contraction-expansion microchannels under electrophoresis

We investigated the dynamics of single DNA molecules driven by the electrophoretic force in several tapered contraction-expansion microchannels. Under high localized electric-field gradients, fast transition between the stretching and compression of DNA molecules was achieved. Numerically, a combination of the finite element method and the coarse-grained Brownian dynamics simulation was used to capture the dynamics of single DNA molecules simplified as freely-draining bead-spring wormlike chains. A generalized predictor-corrector time marching scheme was proposed in this work. It was found that the initial conformation, the initial center-of-mass location, and the electric-field strength are three major factors affecting the DNA dynamics. The forced relaxation due to the reverse compression in the expansion zone can speed the relaxation of DNA molecules compared with the free relaxation in the bulk. We have also simulated DNA dynamics in different contraction-expansion microchannels by changing the length or the small-end width of the contraction zone (with other geometrical lengths fixed). Decreasing the small-end width can provide higher DNA stretching due to both increased Deborah number and increased accumulated strain. Increasing the length of the contraction zone, on the other hand, only slightly increases the accumulated strain, while greatly decreases the Deborah number, causing a decrease in DNA stretching. Experimentally, DNA molecules were gradually stretched in the contraction zone and then were quickly compressed back within a short distance outside the contraction zone. DNA chains in different initial configurations demonstrate different behaviors in contraction-expansion microchannels. The Brownian dynamics simulation results are in qualitative agreement with the experimental observations.

Static conformation and dynamics of single DNA molecules confined in nanoslits

Nearly thirty years ago, Daoud and de Gennes derived the scaling predictions for the linear polymer chains trapped in a slit with dimension close to the Kuhn length; however, these predictions have yet to be compared with experiments. We have fabricated nanoslits with vertical dimension similar to the Kuhn length of ds-DNA (110nm) using standard photolithography techniques. Fluorescently labeled single DNA molecules with contour lengths L ranging from 4 to 75 microm were successfully injected into the slits and the chain molecules undergoing Brownian motions were imaged by fluorescence microscopy. The distributions of the chain radius of gyration and the two-dimensional asphericity were measured. It is found that the DNA molecules exhibit highly anisotropic shape and the mean asphericity is chain length independence. The shape anisotropy of DNA in our measurements is between two and three dimensions (2D and 3D). The static scaling law of the chain extension and the radius of gyration <R parallel>, <Rg> approximately L(nu) were observed with nuR(parallel)=0.65+/-0.02 and nu(Rg)=0.68+/-0.05. These results are close to the average value between two (nuR parallel,Rg=0.75) and three (nuR parallel,Rg=0.6) -dimensional theoretical value. The scaling of the extensional and rotational relaxation time are between Rouse model in nanoslits and Zimm model in the bulk solution, respectively. We show that the conformation and chain relaxation of DNA confined in a slit close to Kuhn length exhibit the quasi-two-dimensional behavior.

High Magnification Microscopic System Focusing on Transparent Liquid/Liquid Interface Formed in Thin-Layer Two-Phase Microcell

A thin-layer two-phase microcell was fabricated without an adhesive, which showed no fluorescence and resisted acids. The lower hollow of the microcell was thin (0.18 mm), which was in the range of the working distance of the used objective of high magnification (60x) and high numerical aperture (1.2). A reflection method using probe light was newly employed for focusing the objective on a transparent dodecane/water interface strictly with an inverted microscope. The system was applied to in situ fluorescence microscopic measurements of the dynamics of single DNA molecules (165600 base pairs) at the dodecane/water interface.

Compaction Dynamics of Single DNA Molecules under Tension

Using transverse magnetic tweezers, we studied the dynamics of DNA compaction induced by hexaammine cobalt chloride under constant forces. Discontinuous DNA compaction events were found for forces ranging from 0.5 to 1.7 pN, with approximately 270 nm DNA adsorbed in each compaction event. Forces larger than 6 pN were found able to unravel the toroid in a similar intermittent stepwise manner. The observations indicate that the folding/unfolding events are transitions between two metastable structural states which are separated by a tension-dependent energy barrier. Analysis of the waiting time revealed that the degree of the package ordering of DNA in a toroid depends on the compaction kinetics.

Statics and Dynamics of Single DNA Molecules Confined in Nanochannels

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics.

Dynamics of DNA conformations and DNA-protein interactions

AbstractOptical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent findings on the search process of proteins for a specific target on the DNA.

Conformation and dynamics of single DNA molecules in parallel-plate slit microchannels

The conformation and diffusion of a single DNA molecule confined between two parallel plates are examined using both single molecule experiments and Brownian dynamics simulations accounting for hydrodynamic interactions. The degree of chain stretching and the diffusivity are characterized as a function of the chain confinement and the channel geometry. Good agreement is found between the simulations, experiments, and scaling theory predictions.

Electrophoretic collision of a DNA molecule with an insulating post.

We study the dynamics of single DNA molecules driven by an electric field into a stationary obstacle. These collisions are broadly classified as "hook" and "roll-off" events. We show that obstacle-induced electric field gradients stretch impacting DNA and thus greatly influence the hooking probability. Consequently, in addition to collision geometry, determination of the hooking probability depends on the Deborah number (De) for 0.5<De<40. Individual DNA impact dynamics are highly configuration sensitive, characteristic of polymers in elongational flows and fields.