DNA Unzipping Research Articles

Shear unzipping of double-stranded DNA

We use a simple nonlinear scaler displacement model to calculate the distribution of effects created by a shear stress on a double-stranded DNA (dsDNA) molecule and the value of shear force F(c) that is required to separate the two strands of a molecule at a given temperature. It is shown that for molecules of base pairs fewer than than 21, the entire single strand moves in the direction of applied force, whereas for molecules having base pairs more than 21, part of the strand moves in the opposite direction under the influence of force acting on the other strand. This result as well as the calculated values of F(c) as a function of length of dsDNA molecules are in very good agreement with the experimental values of Hatch et al. [Phys. Rev. E 78, 011920 (2008)].

Force induced unzipping of DNA with long range correlated noise

We derive and solve a Fokker–Planck equation for the stationary distribution of the freeenergy, in a model of unzipping of double-stranded DNA under external force.The autocorrelation function of the random DNA sequence can be of a generalform, including long range correlations. In the case of Ornstein–Uhlenbeck noise,characterized by a finite correlation length, our result reduces to the exact result ofAllahverdyan et al, with the average number of unzipped base pairs going as⟨X⟩ ∼ 1/f2 in the white noiselimit, where f is the deviation from the critical force. In the case of long range correlatednoise, where the integrated autocorrelation is divergent, we find that⟨X⟩ is finiteat f = 0, with its value decreasing as the correlations become of longer range. This shows that longrange correlations actually stabilize the DNA sequence against unzipping. Our result is alsoin agreement with the findings of Allahverdyan et al obtained using numerical generation ofthe long range correlated noise.

Unzipping of Double-Stranded DNA in Engineered α-Hemolysin Pores

Biological protein α-hemolysin nanopore is under intense investigation as a potential platform for rapid and low-cost DNA sequencing. However, due to its narrow constriction, analysis of DNA in the α-hemolysin pore has long time been restricted to single strands. In this paper, we report that by introducing new surface functional groups into the α-hemolysin pore, facilitated unzipping of double-stranded DNA through the channel could be achieved. Since the mean residence time of the DNA events is dependent on the length of the duplex, and also varies with the nucleotide base composition, the modified protein pore approach offers the potential for rapid double-stranded DNA analysis, including sequencing.

Mechanically Unzipping Double-Stranded DNA with Built-In Sequence Inhomogeneities and Bound Proteins

We theoretically analyze the force signal expected during unzipping of DNA with a) bound proteins and b) when the DNA is more strongly base-paired over certain regions. We consider the case of a single bound protein, multiple isolated bound proteins, bound proteins with cooperative interactions that result in collective force-induced unbinding events, and the case of a very large number of bound proteins. In addition, we also analyze the case where the unzipping proceeds through multiple, isolated DNA regions which are more strongly bound than the surrounding DNA. Our calculations are done in the fixed-extension ensemble. In both cases we find two different types of force traces which we label sawtooth profile and ramp-plateau profile.. In the former, the force extension curve has a series of sawtooth peaks superposed on the usual constant force of ∼15pN found unzipping bare DNA, while in the ramp-plateau case, the force near an unzipping constraint increases roughly linearly and then levels off and then returns to its baseline value (∼15pN) after the protein in unbound or the strongly base-paired region is disrupted allowing the unzipping fork to pass through. These shapes are correlated with the positions of bound proteins or sequence inhomogeneities. We calculate how the force-extension profile depends on the protein or sequence parameters. Our results compare well with observed in unzipping of natural-sequence DNA and DNA with bound proteins. These results point the way toward inferring sequence-related information and protein binding enthalpies from single molecule unzipping experiments.

THE TRANSFER INTEGRAL OPERATOR METHOD IN THE STUDY OF DNA UNZIPPING AND BUBBLE FORMATION

In this work we reexamine the unzipping and bubble formation of DNA in the context of the Peyrard–Bishop–Dauxois DNA model using the transfer integral operator method. After a brief overview of the method, we use it to calculate the probabilities that consecutive base-pairs are stretched beyond a threshold amplitude. We compare the continuous Peyrard–Bishop–Dauxois model with an Ising-type model and demonstrate their similarities. For the Peyrard–Bishop–Dauxois model, we derive an expression for the force that is required to open a double-stranded sequence at one end while the other end is kept closed. In the thermodynamic limit, we use this expression to study the dependence of the unzipping force on the temperature. We also calculate the opening probabilities in the case of zero force and in the case where a force is applied in the middle of the sequence. Analytically, the case where the force is applied in the middle is similar to the case of a homogeneous DNA sequence of strong GC pairs with a defect, in the form of a weaker AT base pair, in the middle. Numerical results verify this similarity.

Force fluctuations assist nanopore unzipping of DNA

We experimentally study the statistical distributions and the voltage dependence of theunzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We thenpropose a quantitative theoretical description considering the nanopore unzipping processas a random walk of the opening fork through the DNA sequence energy landscapebiased by a time-fluctuating force. To achieve quantitative agreement fluctuationsneed to be correlated over the millisecond range and have an amplitude of orderkBT/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that theunzipping process is efficiently enhanced by noise in the kHz range. We further show thatthe unzipping time of short 15 base-pair hairpins does not always increase with theglobal stability of the double helix and we theoretically study the role of DNAelasticity on the conversion of the electrical bias into a mechanical unzipping force.

Force induced melting of the constrained DNA

We develop a simple model to study the effects of the applied force on the melting of a double stranded DNA (dsDNA). Using this model, we could study the stretching, unzipping, rupture and slippagelike transition in a dsDNA. We show that in absence of an applied force, the melting temperature and the melting profile of dsDNA strongly depend on the constrained imposed on the ends of dsDNA. The nature of the phase boundary of the force-temperature diagram, which separates the zipped and the open state for the shearinglike transition is remarkably different than the DNA unzipping.

Drs. Allanore and Vacca reply

<h3>Abstract</h3> Optical traps enable nanoscale manipulation of individual biomolecules while measuring molecular forces and lengths. This ability relies on the sensitive detection of optically trapped particles, typically accomplished using laser-based interferometric methods. Recently, precise and fast image-based particle tracking techniques have garnered increased interest as a potential alternative to laser-based detection, however successful integration of image-based methods into optical trapping instruments for biophysical applications and force measurements has remained elusive. Here we develop a camera-based detection platform that enables exceptionally accurate and precise measurements of biological forces and interactions in a dual optical trap. In demonstration, we stretch and unzip DNA molecules while measuring the relative distances of trapped particles from their trapping centers with sub-nanometer accuracy and precision, a performance level previously only achieved using photodiodes. We then use the DNA unzipping technique to localize bound proteins with extraordinary sub-base-pair precision, revealing how thermal DNA fluctuations allow an unzipping fork to sense and respond to a bound protein prior to a direct encounter. This work significantly advances the capabilities of image tracking in optical traps, providing a state-of-the-art detection method that is accessible, highly flexible, and broadly compatible with diverse experimental substrates and other nanometric techniques.

DNA Translocation and Unzipping through a Nanopore: Some Geometrical Effects

This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric α-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.

Analytical theory of finite-size effects in mechanical desorption of a polymer chain

We discuss a unique system that allows exact analytical investigation of first- and second-order transitions with finite-size effects: mechanical desorption of an ideal lattice polymer chain grafted with one end to a solid substrate with a pulling force applied to the other end. We exploit the analogy with a continuum model and use accurate mapping between the parameters in continuum and lattice descriptions, which leads to a fully analytical partition function as a function of chain length, temperature (or adsorption strength), and pulling force. The adsorption-desorption phase diagram, which gives the critical force as a function of temperature, is nonmonotonic and gives rise to re-entrance. We analyze the chain length dependence of several chain properties (bound fraction, chain extension, and heat capacity) for different cross sections of the phase diagram. Close to the transition a single parameter (the product of the chain length N and the deviation from the transition point) describes all thermodynamic properties. We discuss finite-size effects at the second-order transition (adsorption without force) and at the first-order transition (mechanical desorption). The first-order transition has some unusual features: The heat capacity in the transition region increases anomalously with temperature as a power law, metastable states are completely absent, and instead of a bimodal distribution there is a flat region that becomes more pronounced with increasing chain length. The reason for this anomaly is the absence of an excess surface energy for the boundary between adsorbed and stretched coexisting phases (this boundary is one segment only): The two states strongly fluctuate in the transition point. The relation between mechanical desorption and mechanical unzipping of DNA is discussed.

A Novel Single Molecular Signature for Discriminating DNA Unzipping in a Nanopore

Using nanopore for DNA unzipping has been extensively studied. By measuring the voltage-dependent duration of current blocks produced by the unzipping process, one can evaluate the force and energy involved in the double strands hybridization. However, in the real-time biosensing, other molecules co-existing in the mixture may also produce blocks in similar amplitude and duration. Therefore a characteristic current signature is needed to discriminate the unzipping signal from other blockades. Here we identify a novel molecular signature that can reveal sequential steps in DNA unzipping in the nanopore.

Synergistic Action of RNA Polymerases in Overcoming the Nucleosomal Barrier

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet it must access the nucleosomal DNA. Various lines of in vivo evidence suggest that multiple RNAPs might increase transcription efficiency through nucleosomal DNA. Here we have quantitatively investigated this hypothesis by using E. coli RNAP as a model system and directly monitoring its location on the DNA via a single molecule DNA unzipping technique. When a single RNAP encountered a nucleosome, it paused with a distinctive 10-bp periodicity and was backtracked by an average distance of ∼10-15 bp. When two RNAPs were elongating in close proximity, the trailing RNAP exerted an assisting force on the leading RNAP, reducing its backtracking and enhancing its transcription through a nucleosome ∼5-fold. Taken together, our data indicate that histone-DNA interactions within a nucleosome dictate RNAP pausing behavior, and that alleviation of nucleosome-induced backtracking by multiple polymerases is a likely mechanism for overcoming the nucleosomal barrier in vivo.

Statistical Properties of Metastable Intermediates in DNA Unzipping

We unzip DNA molecules using optical tweezers and determine the sizes of the cooperatively unzipping and zipping regions separating consecutive metastable intermediates along the unzipping pathway. Sizes are found to be distributed following a power law, ranging from one base pair up to more than a hundred base pairs. We find that a large fraction of unzipping regions smaller than 10 bp are seldom detected because of the high compliance of the released single stranded DNA. We show how the compliance of a single nucleotide sets a limit value around 0.1 N/m for the stiffness of any local force probe aiming to discriminate one base pair at a time in DNA unzipping experiments.

The mechanism of DNA mechanical unzipping

The process of DNA double helix unzipping is considered to be a nonlinear dynamical process powered by an external force. In the course of unzipping the most probable pathway of the mechanical strands separation is determined. This pathway accounts for the structural organization of the double helix, the manner in which the external force is applied, and the kinetic parameters of the base pair opening in the double-stranded DNA chain. It is assumed in our model that the base pair unzipping includes the stretch of DNA complementary base pairs and the double helix torsion. For the description of the DNA unzipping the two-stage mechanism of the external force action is proposed. The mechanism explains the cooperative nature of the unzipping process under the physiological conditions and the threshold character of external force action. On the first stage the external force transforms the conformational state of the double helix and makes the unzipping possible. On the second stage the unzipping propagates along dsDNA. The boundary between the open and the closed parts of the helix (the so called ‘fork’) is demonstrated to move along the chain as a step-like excitation (kink soliton). It is shown that for stable unzipping propagation along DNA double helix, it is necessary to allow rotation of the DNA chain while keeping the velocity of the unzipping fork propagation small in comparison to the velocity of sound in the DNA. It is demonstrated that fluctuations of the external force at the constant velocity of unzipping and the fluctuations of the DNA unzipping length at the fixed force have the same origin: the heterogeneity of the DNA base pairs.

Dynamical modeling of molecular constructions and setups for DNA unzipping

We present a dynamical model of DNA mechanical unzipping under the action of a force. The model includes the motion of a fork in a sequence-dependent landscape, the trap(s) acting on the bead(s) and the polymeric components of the molecular construction (unzipped single strands of DNA and linkers). Different setups are considered to test the model, and the outcome of the simulations is compared to simpler dynamical models existing in the literature where polymers are assumed to be at equilibrium.

DNA unzipping phase diagram calculated via replica theory

We show how single-molecule unzipping experiments can provide strong evidence that the zero-force melting transition of long molecules of natural dsDNA should be classified as a phase transition of the higher-order type (continuous). Toward this end, we study a statistical-mechanics model for the fluctuating structure of a long molecule of dsDNA, and compute the equilibrium phase diagram for the experiment in which the molecule is unzipped under applied force. We consider a perfect-matching dsDNA model, in which the loops are volume-excluding chains with arbitrary loop exponent c . We include stacking interactions, hydrogen bonds, and main-chain entropy. We include sequence heterogeneity at the level of random sequences; in particular, there is no correlation in the base-pairing (bp) energy from one sequence position to the next. We present heuristic arguments to demonstrate that the low-temperature macrostate does not exhibit degenerate ergodicity breaking. We use this claim to understand the results of our replica-theoretic calculation of the equilibrium properties of the system. As a function of temperature, we obtain the minimal force at which the molecule separates completely. This critical-force curve is a line in the temperature-force phase diagram that marks the regions where the molecule exists primarily as a double helix versus the region where the molecule exists as two separate strands. We compare our random-sequence model to magnetic tweezer experiments performed on the 48 502 bp genome of bacteriophage lambda . We find good agreement with the experimental data, which is restricted to temperatures between 24 and 50 degrees C . At higher temperatures, the critical-force curve of our random-sequence model is very different for that of the homogeneous-sequence version of our model. For both sequence models, the critical force falls to zero at the melting temperature T_{c} like |T-T_{c}|;{alpha} . For the homogeneous-sequence model, alpha=1/2 almost exactly, while for the random-sequence model, alpha approximately 0.9 . Importantly, the shape of the critical-force curve is connected, via our theory, to the manner in which the helix fraction falls to zero at T_{c} . The helix fraction is the property that is used to classify the melting transition as a type of phase transition. In our calculation, the shape of the critical-force curve holds strong evidence that the zero-force melting transition of long natural dsDNA should be classified as a higher-order (continuous) phase transition. Specifically, the order is 3rd or greater.

Bubble merging in breathing DNA as a vicious walker problem in opposite potentials

We investigate the coalescence of two DNA bubbles initially located at weak domains and separated by a more stable barrier region in a designed construct of double-stranded DNA. In a continuum Fokker-Planck approach, the characteristic time for bubble coalescence and the corresponding distribution are derived, as well as the distribution of coalescence positions along the barrier. Below the melting temperature, we find a Kramers-type barrier crossing behavior, while at high temperatures, the bubble corners perform drift diffusion toward coalescence. In the calculations, we map the bubble dynamics on the problem of two vicious walkers in opposite potentials. We also present a discrete master equation approach to the bubble coalescence problem. Numerical evaluation and stochastic simulation of the master equation show excellent agreement with the results from the continuum approach. Given that the coalesced state is thermodynamically stabilized against a state where only one or a few of the base pairs of the barrier region are re-established, it appears likely that this type of setup could be useful for the quantitative investigation of thermodynamic DNA stability data as well as the rate constants involved in the unzipping and zipping dynamics of DNA in single molecule fluorescence experiments.

Single-molecule Studies of RNA Unzipping Kinetics Using Nanopores

The α-Hemolysin protein pore has been previously used to study the unzipping kinetics of DNA duplexes, taking advantage of its sub-2nm pore constriction, which permits single stranded nucleic acids but not double stranded structures. Here we present the first extensive nanopore study of RNA unzipping kinetics as a function of voltage and temperature, and compare it with DNA unzipping of molecules with identical primary sequence. Our studies reveal clear differences between RNA and DNA, having similar bulk melting properties, highlighting the nanopore ability to probe subtle differences in nucleic acids' free energy and their interactions with the pore. In addition, we find that RNA hybrids with different overhang sequences display large difference in both unzipping times and current blockage signals. These results may be interpreted in the context of strong interactions between poly-A tail and the pore. Our work sheds light on the mechanics of nanopore unzipping and its sensitivity to small differences in nucleic acid base pairing stability. We currently in the process of developing a theoretical model to explain these observations, which will also set the stage for further studies involving multiple hairpin structures of RNA, and to more complicated and interesting biological problems, involving RNA-helicase interactions.1. Mathe, J., Visram, H., Viasnoff, V., Rabin, Y. & Meller, A. (2004) Nanopore unzipping of individual DNA hairpin molecules. Biophys J 87, 3205-3212.2. Dudko, O., Mathe, J., Szabo, A., Meller, A. & Hummer, G. (2007) Extracting kinetics from single-molecule force spectroscopy: Nanopore unzipping of DNA hairpins. Biophys. J. 92, 4188-4195.

Shear Unzipping of DNA

We study theoretically the mechanical failure of a simple model of double-stranded DNA under an applied shear. Starting from a more microscopic Hamiltonian that describes a sheared DNA, we arrive at a nonlinear generalization of a ladder model of shear unzipping proposed earlier by deGennes [de Gennes, P. G. C. R. Acad. Sci., Ser. IV: Phys., Astrophys. 2001, 1505]. Using this model and a combination of analytical and numerical methods, we study the DNA "unzipping" transition when the shearing force exceeds a critical threshold at zero temperature. We also explore the effects of sequence heterogeneity and finite temperature and discuss possible applications to determine the strength of colloidal nanoparticle assemblies functionalized by DNA.

Theoretical Study of Sequence-Dependent Nanopore Unzipping of DNA

We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.