Persistence Length Of DNA Research Articles

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

Fluorescence nanoscopy of single DNA molecules by using stimulated emission depletion (STED).

Temperature Dependence of DNA Persistence Length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5 and 42 C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.

The Mean Looping Time of DNA

In many gene expression systems, a protein located on the DNA can affect the expression of a gene far along the chain. It has been recognized that the DNA can form transient loops, bringing a specific region of the gene close to another. Thus, transcription can be activated when a transcription factor is positioned far away from its site. The frequency of bending is a characteristic time scale of the activation process.The mean time for a DNA molecule to loop, bringing together two sites, is a fundamental factor that we studied. Various approximations have been used to model polymers. Interestingly, dsDNA has been found to be well described by the standard Rouse model, in which the polymer is described as a collection of bead monomers connected by harmonic springs. The Rouse model is relevant when the sites are at a distance considerably bigger than the DNA persistence length. When the distance between the sites is of several persistence lengths, the semi-flexible chain model is better suited to model the DNA dynamics. The polymer chain is subjected to random independent motion (Brownian motion). When the two monomers come closer than a certain distance, interaction takes place and the monomers connect. We assumed that the interaction rate is much faster than the encounter time, thus the process ends with the first encounter of the monomers. This allowed us to compute the asymptotic formula for the mean encounter time in the two models. We obtained precise estimates for this mean first encounter time in two and three dimensions. Brownian simulations confirm our formulas and we discuss consequences of our results for random gene activation in the nucleus.

Freely Orbiting Magnetic Tweezers: A New Twist on Single Molecule Force Spectroscopy

A wide variety of cellular processes, including replication, transcription, and recombination, alter the linking number of DNA. Understanding such processes therefore requires measuring rotation; however, it has only recently become possible to make such measurements directly at the single molecule scale. We have developed a new instrument, the freely orbiting magnetic tweezer (FOMT), which permits straightforward, direct measurement of rotary motion in biopolymers.We demonstrate that FOMT can observe rotation of a dsDNA tether simply by tracking the trajectory of an unlabeled, commercially available, superparamagnetic bead. We show that no torque is applied to the system by the magnet, facilitating unbiased studies of processes that untwist dsDNA over many turns. Further, we demonstrate that, similar to a conventional magnetic tweezer, controlled forces may be applied to the complex under study.We also discuss results from experiments employing FOMT, including including measurements of the torsional persistence length of DNA in the 0 - 2.5 pN force range, and direct observation of DNA unwinding during RecA filament formation.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Sequence Dependence of DNA Persistence Length

It is vital for many aspects of DNA-protein interaction to know how DNA bending rigidity depends on its sequence. In particular, this is important for understanding how nucleosomes position along DNA molecules. Although the problem has been discussed for decades, it remains unsolved. The main difficulty is that one has to measure the rigidity constants for DNA molecules of different sequences with very high accuracy. The only known method that provides the needed accuracy is based on cyclization of short DNA fragments. Here we used the method to find how DNA bending rigidity depends on its sequence. We addressed the problem using the dinucleotide approximation, in which particular values of rigidity constants are assigned to each of 10 distinct dinucleotide steps. We prepared DNA fragments, about 200 bp in length, with various quasi-periodic sequences, measured their cyclization efficiency, and fitted the data by a theoretical equation to obtain the bending rigidity constant (or persistence length). By combining the data for all fragments we were able to extract a full set of rigidity constants. To test the resulting set of constants we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments and determined their values of a experimentally. We obtained remarkably close agreement between the measured and calculated values of a, proving that we have found the correct solution of this long-standing problem. This result opens new opportunities to test different models of sequence specificity of DNA-protein interaction.

Conformational analysis and estimation of the persistence length of DNA using atomic force microscopy in solution

The worm-like chain model describes the mechanical properties of semi-flexible polymers by introducing a certain correlation length along the contour. This correlation length is called the persistence length. Using atomic force microscopy in solution, we performed measurements of the persistence length of DNA molecules. We found good agreement between the theoretical model and experimental data. However, the measured persistence length values in solution differ from those found by several authors using the same technique but in dry air. In order to determine the contribution of the electrostatic persistence length to the total persistence length, we varied the salt concentration. We found a large discrepancy between the Odijk, Skolnick and Fixman theory and our measurements. The effect of divalent ions and the omission in the theory of the dependence of non-electrostatic persistence length on salt concentration are qualitatively invoked.

Ethanol induces condensation of single DNA molecules

As a widely used precipitation agent for DNA extraction, ethanol is used to induce single molecule DNA condensation. This process is studied with force-measuring magnetic tweezers and atomic force microscopy (AFM). Our experiments provide direct evidence of the metastable intermediate racquet states in DNA collapse induced by ethanol. The measured condensing force is less than 0.2 pN even at 50% ethanol concentration, which is much less than those induced by multivalent cations and cationic surfactants. We confirmed the A-B transition of DNA in ethanol and found that the tensile modulus of A-form DNA is larger than that of B-form. The single molecule pulling experiment shows very different features of neutral ethanol from those of multivalent cations. The pulling curve contains a wide range of step sizes, ranging from tens of nanometres to a few micrometres, contrasting with the relatively uniform interval (about 200 nm) in multivalent cations. Meanwhile, the persistence length of DNA decreases monotonically with the increasing ethanol concentration. The condensing morphologies by the weak attraction of DNA segments in the less polar solvent are loose and flowerlike structures composed of many annealed irregular racquets. The analysis of pulling experiments is supported by AFM direct imaging. We concluded that the dominant factor in DNA condensation induced by ethanol is solvent exclusion rather than the charge neutralization correlation effect.

DNA Strands Attached Inside Single Conical Nanopores: Ionic Pore Characteristics and Insight into DNA Biophysics

Single nanopores attract a great deal of scientific interest as a basis for biosensors and as a system to study the interactions and behavior of molecules in a confined volume. Tuning the geometry and surface chemistry of nanopores helps create devices that control transport of ions and molecules in solution. Here, we present single conically shaped nanopores whose narrow opening of 8 or 12 nm is modified with single-stranded DNA molecules. We find that the DNA occludes the narrow opening of nanopores and that the blockade extent decreases with the ionic strength of the background electrolyte. The results are explained by the ionic strength dependence of the persistence length of DNA. At low KCl concentrations (10 mM) the molecules assume an extended and rigid conformation, thereby blocking the pore lumen and reducing the flow of ionic current to a greater extent than compacted DNA at high salt concentrations. Attaching flexible polymers to the pore walls hence creates a system with tunable opening diameters in order to regulate transport of both neutral and charged species.

Chemically accurate coarse graining of double-stranded DNA.

Coarse-grained (CG) modeling approaches are widely used to simulate many important biological processes involving DNA, including chromatin folding and genomic packaging. The bending propensity of a semiflexible DNA molecule critically influences these processes. However, existing CG DNA models do not retain a sufficient fidelity of the important local chain motions, whose propagation at larger length scales would generate correct DNA persistent lengths, in particular when the solution's ionic strength is widely varied. Here we report on a development of an accurate CG model for the double-stranded DNA chain, with explicit treatment of mobile ions, derived systematically from all-atom molecular dynamics simulations. Our model generates complex local motions of the DNA chain, similar to fully atomistic dynamics, leading also to a quantitative agreement of our simulation results with the experimental data on the dependence of the DNA persistence length on the solution ionic strength. We also predict a structural transition in a torsionally stressed DNA nanocircle as the buffer ionic strength is increased beyond a threshold value.

Temperature dependence of DNA persistence length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5°C and 42°C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.

Anharmonic Torsional Stiffness of DNA Revealed under Small External Torques

DNA supercoiling plays an important role in a variety of cellular processes. The torsional stress related to supercoiling may also be involved in gene regulation through the local structure and dynamics of the double helix. To check this possibility, steady torsional stress was applied in the course of all-atom molecular dynamics simulations of two DNA fragments with different base pair sequences. For one fragment, the torsional stiffness significantly varied with small twisting. The effect is traced to sequence-specific asymmetry of local torsional fluctuations, and it should be small in long random DNA due to compensation. In contrast, the stiffness of special short sequences can change significantly, which gives a simple possibility of gene regulation via probabilities of strong fluctuations. These results have important implications for the role of DNA twisting in complexes with transcription factors.

Dielectrophoresis of DNA: Quantification by impedance measurements.

Dielectrophoretic properties of DNA have been determined by measuring capacitance changes between planar microelectrodes. DNA sizes ranged from 100 bp to 48 kbp, DNA concentrations from below 0.1 to 70 mugml. Dielectrophoretic spectra exhibited maximum response around 3 kHz and 3 MHz. The strongest response was found for very long DNA (above 10 kbp) and for short 100 bp fragments, which corresponds to the persistence length of DNA. The method allows for an uncomplicated, automatic acquisition of the dielectrophoretic properties of submicroscopical objects without the need for labeling protocols or optical accessibility.

Mechanical and structural properties of YOYO-1 complexed DNA

YOYO-1 is a fluorescent dye widely used for probing the statistical–mechanical properties of DNA. However, currently contradicting data exist how YOYO-1 binding alters the DNA structure and rigidity. Here, we systematically address this problem using magnetic tweezers. Remarkably, we find that the persistence length of DNA remains constant independent of the amount of bound YOYO-1, which contrasts previous assumptions. While the ionic conditions can considerably alter the stability of YOYO-1 binding, the DNA bending rigidity seems not to be affected. We furthermore determine important structural parameters such as the binding site size, the elongation, as well as the untwisting angle per bound YOYO-1 molecule. We expect that our assay, in which all the parameters are determined within a single experiment, will be beneficial for a large range of other DNA binding drugs.

Effect of Genomic Long-Range Correlations on DNA Persistence Length: From Theory to Single Molecule Experiments

Sequence dependency of DNA intrinsic bending properties has been emphasized as a possible key ingredient to in vivo chromatin organization. We use atomic force microscopy (AFM) in air and liquid to image intrinsically straight (synthetic), uncorrelated (hepatitis C RNA virus) and persistent long-range correlated (human) DNA fragments in various ionic conditions such that the molecules freely equilibrate on the mica surface before being captured in a particular conformation. 2D thermodynamic equilibrium is experimentally verified by a detailed statistical analysis of the Gaussian nature of the DNA bend angle fluctuations. We show that the worm-like chain (WLC) model, commonly used to describe the average conformation of long semiflexible polymers, reproduces remarkably well the persistence length estimates for the first two molecules as consistently obtained from (i) mean square end-to-end distance measurement and (ii) mean projection of the end-to-end vector on the initial orientation. Whatever the operating conditions (air or liquid, concentration of metal cations Mg(2+) and/or Ni(2+)), the persistence length found for the uncorrelated viral DNA underestimates the value obtained for the straight DNA. We show that this systematic difference is the signature of the presence of an uncorrelated structural intrinsic disorder in the hepatitis C virus (HCV) DNA fragment that superimposes on local curvatures induced by thermal fluctuations and that only the entropic disorder depends upon experimental conditions. In contrast, the WLC model fails to describe the human DNA conformations. We use a mean-field extension of the WLC model to account for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of human genomic DNA: the stronger the LRC, the smaller the persistence length. The comparison of AFM imaging of human DNA with LRC DNA simulations confirms that the rather small mean square end-to-end distance observed, particularly for G+C-rich human DNA molecules, more likely results from a large-scale intrinsic curvature due to a persistent distribution of DNA curvature sites than from some increased flexibility.

Persistence length of DNA molecules confined in nanochannels

The influence of confinement on the persistence length of dsDNA molecules under a high ionic strength environment was explored by coarse-grained Monte Carlo simulations in channels of different profiles. It was found that under confinement three definitions of the persistence length of DNA molecules were not equivalent and represented different properties. In case of the global quantities, the projection and the WLC persistence lengths, the apparent values up to several hundred nanometres are observed for DNA confined in narrow channels. The orientational correlation function cos theta(s) of confined DNA shows a complex pattern, distinctive for semiflexible polymers. At weak and moderate confinements the function cos theta(s) suggests an unexpected increase in the apparent DNA flexibility. The orientational persistence length computed from the initial slope of the function cos theta(s) mirrors only short-scale correlations and gives the value close to the intrinsic persistence length of DNA. The simulation data of direct relevance to experimental studies of DNA in microfluidic devices are compared with analytical theories for stiff chains.

Ethanol Induced Shortening of dsDNA in Nanochannels

The entropic confinement and manipulation of DNA in fabricated nanostructures has facilitated both the study of DNA-protein interactions and the polymer physics of DNA conformations in different solvent conditions and geometries. Moreover, it holds great promise as a powerful tool for rapid genomic sequencing. Ethanol precipitation is a common tool in molecular biology used to purify and concentrate DNA, typically in 70% (or greater) ethanol solutions. Even at lower ethanol concentrations, however, DNA has been shown to undergo a transformation from its physiological B-form to A-form, a shorter yet slightly less twisted molecular conformation. To examine this transition, we isolated individual YOYO-1 labeled λ-DNA molecules in 100nm×100nm nanochannels in 0, 20, 40 and 60% ethanol solutions. We observed a dramatic shortening in the mean measured lengths with increasing ethanol and a broadening of the distribution of measured lengths at the intermediate ethanol concentrations. These observed lengths are less than that of fully A-form λ-DNA, suggesting that other mechanisms are involved in shortening the observed molecules. First, the possible effect of ethanol dislodging of the intercolated fluorophores and subsequent shortening the observed molecule is discussed. Second, the substantial variations in intensity in our observed molecules at the higher ethanol concentrations are (i) suggestive of the higher order DNA conformations such as loops and toroids that have been observed in DNA dried on mica surfaces and (ii) in accord with the observation that the effective persistence length of DNA is reduced in ethanol solutions.

Persistence length and scaling properties of single-stranded DNA adsorbed on modified graphite

We have characterized the polymer physics of single-stranded DNA (ssDNA) using atomic force microscopy. The persistence length l(p) of circular ssDNA adsorbed on a modified graphite surface was determined independently of secondary structure. At a very low ionic strength we obtained l(p)=9.1 nm from the bond correlation function. Increasing the salt concentration lead to a decrease in l(p); at 1 mM NaCl we found l(p)=6.7 nm, while at 10 mM NaCl a value l(p)=4.6 nm was obtained. The persistence length was also extracted from the root-mean-square end-to-end distance and the end-to-end distance distribution function. Finally, we have investigated the scaling behavior using the two latter quantities, and found that on long length scales ssDNA behaves as a two-dimensional self-avoiding walk.

DNA Deformations near Charged Surfaces: Electron and Atomic Force Microscopy Views

DNA is a very important cell structural element, which determines the level of expression of genes by virtue of its interaction with regulatory proteins. We use electron (EM) and atomic force microscopy (AFM) to characterize the flexibility of double-stranded DNA (∼150–950 nm long) close to a charged surface. Automated procedures for the extraction of DNA contours (∼10–120 nm for EM data and ∼10–300 nm for AFM data) combined with new statistical chain descriptors indicate a uniquely two-dimensional equilibration of the molecules on the substrate surface regardless of the procedure of molecule mounting. However, in contrast to AFM, the EM mounting leads to a noticeable decrease in DNA persistence length together with decreased kurtosis. Analysis of local bending on short length scales (down to 6 nm in the EM study) shows that DNA flexibility behaves as predicted by the wormlike chain model. We therefore argue that adhesion of DNA to a charged surface may lead to additional static bending (kinking) of ∼5 degrees per dinucleotide step without impairing the dynamic behavior of the DNA backbone. Implications of this finding are discussed.

Model systems for DNA and its environment: Suitability and accuracy in theoretical calculations

An investigation of the effects of different approximations on the results of Poisson-Boltzmann and Monte Carlo calculations of the accumulation of counterions at the highly charged surface of DNA is presented. Uniformly charged cylinders of various finite lengths are used to model a segment of DNA and the results of Poisson-Boltzmann calculations are compared with those obtained using an infinitely long charged cylinder. Results of Poisson-Boltzmann calculations using all-atom models of different lengths are also compared in a study of end effects. The use of rigid structures that are significantly shorter than is the persistence length of DNA appears to be justified. Monte Carlo calculations of counterion densities around charged cylinder models have been performed to assess the magnitude of the statistical errors in this approach. Comparison of the calculated local densities at different, symmetrically equivalent, positions indicate that even after 20 million configurations have been sampled there is a small but significant error in the concentrations calculated over regions with a volume of 3.36 {angstrom}{sup 3}. Monte Carlo calculations for 1 and 4 {angstrom} hard-sphere counterions are presented with results given after 5 million, 10 million, and 20 million configurations were sampled. The results of these calculations show amore » strong dependence on the model system used and demonstrate the need for accurate geometric details in the calculations.« less

The persistence length of double stranded DNA determined using dark field tethered particle motion

The wormlike chain model describes the micromechanics of semiflexible polymers by introducing the persistence length. We propose a method of measuring the persistence length of DNA in a controllable near-native environment. Using a dark field microscope, the projected positions of a gold nanoparticle undergoing constrained Brownian motion are captured. The nanoparticle is tethered to a substrate using a single double stranded DNA (dsDNA) molecule and immersed in buffer. No force is exerted on the DNA. We carried out Monte Carlo simulations of the experiment, which give insight into the micromechanics of the DNA and can be used to interpret the motion of the nanoparticle. Our simulations and experiments demonstrate that, unlike other similar experiments, the use of nanometer instead of micrometer sized particles causes particle-substrate and particle-DNA interactions to be of negligible effect on the position distribution of the particle. We also show that the persistence length of the tethering DNA can be estimated with a statistical error of 2 nm, by comparing the statistics of the projected position distribution of the nanoparticle to the Monte Carlo simulations. The persistence lengths of 45 single molecules of four different lengths of dsDNA were measured under the same environmental conditions at high salt concentration. The persistence lengths we found had a mean value of 35 nm (standard error of 2.8 nm), which compares well to previously found values using similar salt concentrations. Our method can be used to directly study the effect of the environmental conditions (e.g., buffer and temperature) on the persistence length.