DNA Contour Length Research Articles

Detection of Single DNA Molecule Hybridization on a Surface by Atomic Force Microscopy

Improving the detection of DNA hybridization is a critical issue for several challenging applications encountered in microarray and biosensor domains. Herein, it is demonstrated that hybridization between complementary single-stranded DNA (ssDNA) molecules loosely adsorbed on a mica surface can be achieved thanks to fine-tuning of the composition of the hybridization buffer. Single-molecule DNA hybridization occurs in only a few minutes upon encounters of freely diffusing complementary strands on the mica surface. Interestingly, the specific hybridization between complementary ssDNA is not altered in the presence of large amounts of nonrelated DNA. The detection of single-molecule DNA hybridization events is performed by measuring the contour length of DNA in atomic force microscopy images. Besides the advantage provided by facilitated diffusion, which promotes hybridization between probes and targets on mica, the present approach also allows the detection of single isolated DNA duplexes and thus requires a very low amount of both probe and target molecules.

Thermoadsorptive separation of DNA by size using a polymeric sorbent

The article describes theoretical aspects of isolation and separation of DNA fragments by size using a polymeric sorbent. On this basis we presented a theoretical model of thermal desorption of DNA fragments of different length. Assuming that, under certain pH, interaction of DNA with the polymer sorbent is of an ion-dipole character and moving dipoles interact with the sorbent according to the Boltzmann distribution with the total charge of the DNA fragment in its pore, the expression for the interaction energy as a function of temperature is obtained. Equating the thermal energy to the interaction energy of the ion-dipole character, we have found the critical temperature of DNA separation from the sorbent. Account of the conformation of single-stranded DNA in the coil form leads to the dependence of DNA separation temperature, i.e., desorption, on the length of the DNA chain. The temperature for desorption increases symbatically to the contour length of DNA.

Knotting of linear DNA in nano-slits and nano-channels: a numerical study.

The amount and type of self-entanglement of DNA filaments is significantly affected by spatial confinement, which is ubiquitous in biological systems. Motivated by recent advancements in single DNA molecule experiments based on nanofluidic devices and by the introduction of algorithms capable of detecting knots in open chains, we investigate numerically the entanglement of linear, open DNA chains confined inside nano-slits. The results regard the abundance, type, and length of occurring knots and are compared with recent findings for DNA inside nano-channels. In both cases, the width of the confining region, D, spans the 30 nm-1 μm range and the confined DNA chains are 1-4 μm long. It is found that the knotting probability is maximum for slit widths in the 70-100 nm range. However, over the considered DNA contour lengths, the maximum incidence of knots remains below 20%, while for channel confinement it tops 50%. Further differences of the entanglement are seen for the average contour length of the knotted region, which drops significantly below D ~100 nm for channel-confinement, while it stays approximately constant for slit-like confinement. These properties ought to reverberate in different kinetic properties of linear DNA depending on confinement and could be detectable experimentally or exploitable in nano-technological applications.

Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.

80 A new approach in determining the rigidity of nucleic acids and polymers

In polymer physics, persistence length of the chain molecules varying in degrees of stiffness is usually evaluated from hydrodynamic data for a set of polymers of different molecular mass М. It is known that from the extrapolation of translational diffusion coefficient to infinite molecular mass, one can calculate persistence length and hydrodynamic radius for rigid and semi-rigid molecules. If the persistence length of the molecule is much greater than its contour length, then the hydrodynamic parameters of the chain are independent of its size and such extrapolation is not applicable. In this case we proposed to plot the dependence M/s 0 2 (s 0 – sedimentation coefficient) or MD 2 (D – translational diffusion coefficient) vs. M (in the absence of volume effects) or n (number of monomers). In these coordinates the appearance of a plateau will correspond to the complete impermeability of the molecule and from plateau height the persistence length can be calculated. We analyzed literature data for a few synthetic polymers by our approach and determined their persistence lengths, the values of which were very close to those in the literature. Usually, the synthesis of polymer molecules with very high molecular mass is a very difficult task. But at the same time, natural polymers with high molecular mass are common in biology. A typical example is a DNA molecule. With increasing contour length of DNA, its conformation passes the next stages: (1) a rigid rod-like particle, (2) a semi-rigid permeable coil, and (3) an impermeable Gaussian coil (Serdyuk, Zaccai, & Zaccai, 2007). We analyzed by our approach sedimentation data of DNA molecules from the literature. It was shown that the plateau starts from 40,000 base pairs of the DNA molecule and the estimated value of persistence length was about 50 nm. This value is close to that from literature data (Lu, Weers, & Stellwagen, 2002). Thus, the proposed approach can be applied for flexibility analysis of biological macromolecules of different natures – from nucleic acids to proteins in strong denaturants.

Synthesis and DNA interaction of ethylenediamine platinum(II) complexes linked to DNA intercalants

A series of ethylenediamine platinum(II) complexes connected through semi-rigid chains of 1,2-bis(4-pyridyl)ethane to DNA intercalating subunits (naphthalene, anthracene or phenazine) has been synthesized, and their interactions with calf thymus (CT) DNA have been evaluated by viscometric titrations and equilibrium dialysis experiments. The parent ligands that contain anthracene or phenazine chromophores showed a monointercalative mode of DNA interaction (especially the anthracene derivative), with apparent association constants in the order of 104M−1. The corresponding platinum(II) complexes bind CT DNA through bisintercalation, as established by the significant increase of DNA contour length inferred from viscosity measurements, and the association constants are in the order of 105M−1. The naphthalene derivatives, however, exhibit a mixed mode of interaction, which suggests a partial contribution of both intercalation and groove binding for the ligand, and monointercalation in the case of the platinum(II) complex. Competition dialysis experiments carried out on the intercalative compounds have revealed a moderate selectivity towards GC DNA sequences for the derivatives containing the anthracene chromophore.

Mesoscopic models for DNA stretching under force: New results and comparison with experiments

Single-molecule experiments on double-stranded B-DNA stretching have revealed one or two structural transitions, when increasing the external force. They are characterized by a sudden increase of DNA contour length and a decrease of the bending rigidity. The nature and the critical forces of these transitions depend on DNA base sequence, loading rate, salt conditions and temperature. It has been proposed that the first transition, at forces of 60-80 pN, is a transition from B to S-DNA, viewed as a stretched duplex DNA, while the second one, at stronger forces, is a strand peeling resulting in single-stranded DNAs (ssDNA), similar to thermal denaturation. But due to experimental conditions these two transitions can overlap, for instance for poly(dA-dT). In an attempt to propose a coherent picture compatible with this variety of experimental observations, we derive an analytical formula using a coupled discrete worm-like chain-Ising model. Our model takes into account bending rigidity, discreteness of the chain, linear and non-linear (for ssDNA) bond stretching. In the limit of zero force, this model simplifies into a coupled model already developed by us for studying thermal DNA melting, establishing a connection with previous fitting parameter values for denaturation profiles. Our results are summarized as follows: i) ssDNA is fitted, using an analytical formula, over a nano-Newton range with only three free parameters, the contour length, the bending modulus and the monomer size; ii) a surprisingly good fit on this force range is possible only by choosing a monomer size of 0.2 nm, almost 4 times smaller than the ssDNA nucleobase length; iii) mesoscopic models are not able to fit B to ssDNA (or S to ss) transitions; iv) an analytical formula for fitting B to S transitions is derived in the strong force approximation and for long DNAs, which is in excellent agreement with exact transfer matrix calculations; v) this formula fits perfectly well poly(dG-dC) and λ-DNA force-extension curves with consistent parameter values; vi) a coherent picture, where S to ssDNA transitions are much more sensitive to base-pair sequence than the B to S one, emerges. This relatively simple model might allow one to further study quantitatively the influence of salt concentration and base-pairing interactions on DNA force-induced transitions.

Relation between the change in DNA elasticity on ligand binding and the binding energetics

The widespread use of tweezers for measurement of ligand-DNA binding parameters is based on the McGhee-von Hippel treatment of the DNA contour and persistence length as a function of concentration. The McGhee-von Hippel approach contains the basic assumption that the binding constant K is independent of the number of already bound ligands. However, the change in elasticity of DNA on binding affects the entropic part of the Gibbs free energy and, hence, the K value in a concentration-dependent manner, making the whole approach inconsistent. In the present work we show that the energetic effect of DNA stiffening on noncovalent binding of small ligands is negligible with respect to the net energy of reaction, whereas the DNA stiffening on binding of large ligands must always be considered in each particular case.

Oligomerization and DNA binding of Ler, a master regulator of pathogenicity of enterohemorrhagic and enteropathogenic Escherichia coli

Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 3–5 units of Ler. DNA wraps around Ler particles that contain more than 15–16 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.

Two distinct overstretched DNA structures revealed by single-molecule thermodynamics measurements

Double-stranded DNA is a dynamic molecule whose structure can change depending on conditions. While there is consensus in the literature about many structures DNA can have, the state of highly-stretched DNA is still not clear. Several groups have shown that DNA in the torsion-unconstrained B-form undergoes an "overstretching" transition at a stretching force of around 65pN, which leads to approximately 1.7-fold elongation of the DNA contour length. Recent experiments have revealed that two distinct structural transitions are involved in the overstretching process: (i) a hysteretic "peeling" off one strand from its complementary strand, and (ii) a nonhysteretic transition that leads to an undetermined DNA structure. We report the first simultaneous determination of the entropy (ΔS) and enthalpy changes (ΔH) pertaining to these respective transitions. For the hysteretic peeling transition, we determined ΔS∼20cal/(K.mol) and ΔH∼7kcal/mol. In the case of the nonhysteretic transition, ΔS∼-3cal/(K.mol) and ΔH∼1kcal/mol. Furthermore, the response of the transition force to salt concentration implies that the two DNA strands are spatially separated after the hysteretic peeling transition. In contrast, the corresponding response after the nonhysteretic transition indicated that the strands remained in close proximity. The selection between the two transitions depends on DNA base-pair stability, and it can be illustrated by a multidimensional phase diagram. Our results provide important insights into the thermodynamics of DNA overstretching and conformational structures of overstretched DNA that may play an important role in vivo.

Probing DNA conformational changes with high temporal resolution by tethered particle motion

The tethered particle motion (TPM) technique informs about conformational changes of DNA molecules, e.g. upon looping or interaction with proteins, by tracking the Brownian motion of a particle probe tethered to a surface by a single DNA molecule and detecting changes of its amplitude of movement. We discuss in this context the time resolution of TPM, which strongly depends on the particle–DNA complex relaxation time, i.e. the characteristic time it takes to explore its configuration space by diffusion. By comparing theory, simulations and experiments, we propose a calibration of TPM at the dynamical level: we analyze how the relaxation time grows with both DNA contour length (from 401 to 2080 base pairs) and particle radius (from 20 to 150 nm). Notably we demonstrate that, for a particle of radius 20 nm or less, the hydrodynamic friction induced by the particle and the surface does not significantly slow down the DNA. This enables us to determine the optimal time resolution of TPM in distinct experimental contexts which can be as short as 20 ms.

Revisiting the neighbor exclusion model and its applications

We review the neighbor exclusion model and some of its applications to analyze the binding data of DNA-ligand complexes. We revisit the closed form of the model developed by McGhee and von Hippel in 1974, showing that this classic model can be used to help studying the behavior of DNA contour and persistence lengths when interacting with intercalating ligands. We present methods to quantitatively analyze the variation of these two quantities, allowing one to determine important parameters of the interaction such as the intrinsic binding constant and the exclusion number of the ligand.

A simple and optimized length estimator for digitized DNA contours

The determination of the contour length of DNA imaged by either electron microscopy or atomic force microscopy is frequently required for investigating the physical properties of nucleic acids. Nevertheless, these measurements are often carried out with methods that are not optimized for the curvilinear shape of DNA or are too complex to be of practical use. The aim of this study is to provide a method for the contour length measurements of DNA that is accurate, practical, and computationally simple. Computer simulated DNA fragments were used as experimental benchmarks in order to compute the coefficients a and b of the (n(e), n(o))-characterization [L(n(e),n(o)) = an(e) + bn(o)] so as to minimize the error of the measurements. The data show that, at variance with straight lines, a DNA length estimator depends on both the DNA flexibility and the image resolution, but it is independent of the DNA contour length. A table with DNA estimators to be used for length measurements of digitized contours obtained under commonly used imaging conditions is provided. Although the method has been developed using DNA as a benchmark, its applicability can be extended to other polymers as well as to other imaging techniques.

Entropic Unfolding of DNA Molecules in Nanofluidic Channels

Single DNA molecules confined to nanoscale fluidic channels extend along the channel axis in order to minimize their conformational free energy. When such molecules are forced into a nanoscale fluidic channel under the application of an external electric field, monomers near the middle of the DNA molecule may enter first, resulting in a folded configuration with less entropy than an unfolded molecule. The increased free energy of a folded molecule results in two effects: an increase in extension factor per unit length for each segment of the molecule, and a spatially localized force that causes the molecule to spontaneously unfold. The ratio of this unfolding force to hydrodynamic friction per DNA contour length is measured in nanochannels with two different diameters.

Effects of electrostatic screening on the conformation of single DNA molecules confined in a nanochannel

Single T4-DNA molecules were confined in rectangular-shaped channels with a depth of 300 nm and a width in the range of 150-300 nm casted in a poly(dimethylsiloxane) nanofluidic chip. The extensions of the DNA molecules were measured with fluorescence microscopy as a function of the ionic strength and composition of the buffer as well as the DNA intercalation level by the YOYO-1 dye. The data were interpreted with the scaling theory for a wormlike polymer in good solvent, including the effects of confinement, charge, and self-avoidance. It was found that the elongation of the DNA molecules with decreasing ionic strength can be interpreted in terms of an increase of the persistence length. Self-avoidance effects on the extension are moderate, due to the small correlation length imposed by the channel cross-sectional diameter. Intercalation of the dye results in an increase of the DNA contour length and a partial neutralization of the DNA charge, but besides effects of electrostatic origin it has no significant effect on the bare bending rigidity. In the presence of divalent cations, the DNA molecules were observed to contract, but they do not collapse into a condensed structure. It is proposed that this contraction results from a divalent counterion mediated attractive force between the segments of the DNA molecule.

Geometry of Mediating Protein Affects the Probability of Loop Formation in DNA

Recent single molecule experiments have determined the probability of loop formation in DNA as a function of the DNA contour length for different types of looping proteins. The optimal contour length for loop formation as well as the probability density functions have been found to be strongly dependent on the type of looping protein used. We show, using Monte Carlo simulations and analytical calculations, that these observations can be replicated using the wormlike-chain model for double-stranded DNA if we account for the nonzero size of the looping protein. The simulations have been performed in two dimensions so that bending is the only mode of deformation available to the DNA while the geometry of the looping protein enters through a single variable which is representative of its size. We observe two important effects that seem to directly depend on the size of the enzyme: 1), the overall propensity of loop formation at any given value of the DNA contour length increases with the size of the enzyme; and 2), the contour length corresponding to the first peak as well as the first well in the probability density functions increases with the size of the enzyme. Additionally, the eigenmodes of the fluctuating shape of the looped DNA calculated from simulations and theory are in excellent agreement, and reveal that most of the fluctuations in the DNA occur in regions of low curvature.

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA–dT).poly(dA–dT), poly(dG).poly(dC) and poly(dG–dC).poly(dG–dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT) > poly(dA–dT).poly(dA–dT) > poly(dG–dC).poly(dG–dC) > poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r = 0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding–rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ∼10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.

Ss-LrpB from Sulfolobus solfataricus Condenses about 100 Base Pairs of Its Own Operator DNA into Globular Nucleoprotein Complexes

Ss-LrpB from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2 is a member of the Lrp-like family of Bacterial/Archaeal transcription regulators that binds its own control region at three regularly spaced and partially conserved 15-bp-long imperfect palindromes. We have used atomic force microscopy to analyze the architecture of Ss-LrpB.DNA complexes with a different stoichiometry formed with the wild type operator and with an operator mutant. Binding of dimeric Ss-LrpB to all three target sites is accompanied by the formation of globular complexes, in which the protein induces strong DNA deformations. Furthermore, DNA contour length foreshortening of these complexes indicates DNA wrapping, with about 100 bp being condensed. The average bending angle is 260 degrees . The establishment of protein-protein contacts between Ss-LrpB dimers in these globular complexes will contribute to the cooperativity of the binding. The profound remodeling of the control region is expected to have a strong impact on gene expression and might constitute the key element in the autoregulatory process.

Amine terminated G-6 PAMAM dendrimer and its interaction with DNA probed by Hoechst 33258

Fluorescence characteristics of Hoechst 33258 bound to G-6 dendrimer, to the DNA–G-6 dendrimer complex, and to DNA were compared with that in an aqueous solution. The spectral properties including fluorescence emission spectrum, accessibility of anionic quencher, as well as the fluorescence decay time of the Hoechst 33258 are different for all three conditions, indicating that the environments in these conditions are different. Close analysis of the fluorescence properties led us to suggest that Hoechst 33258 located at or near the contact area of the dendrimer and DNA in the DNA–G-6 complex. In the complex, in the absence of Hoechst 33258, the shape of the circular dichroism in the DNA absorption region remained, indicating that DNA is in B form in the complex. On the other hand, the magnitude of linear dichroism (LD) decreased upon DNA–G-6 dendrimer complex formation. The decrease in LD magnitude reflects the shortening of the DNA contour length, which is expected from the fact that a large part of linear DNA is required to wrap the surface of G-6 dendrimer.