DNA Melts Research Articles

Force induced DNA melting in the presence of an attractive surface.

The self avoiding walk (SAW) model of the polymer has been extended to study the equilibrium properties of double stranded DNA (dsDNA) where two strands of the dsDNA are modeled by two mutually attracting self-avoiding walks (MASAWs) in the presence of an attractive surface. We study simultaneous adsorption and force induced melting transitions and explore different phases of DNA. It is observed that melting is entropically dominated, which can be substantially reduced under the application of an applied force. We consider three scenarios, where the surface is weakly, moderately and highly attractive. For both weakly and moderately attractive surfaces, the DNA desorbs from the surface in a zipped form and acquires the conformation of a melted state with the rise in temperature. However, for a strongly attractive surface, the force applied at one end of the strand (strand-II) results in unzipping, while the other strand (strand-I) remains adsorbed on the surface. We identify this as adsorption-induced unzipping, where the force applied on a single strand (strand-II) can unzip the dsDNA if the surface interaction energy exceeds a specific threshold. We also note that at a moderate surface attraction, the desorbed-zipped DNA melts with an increase in temperature and the free strand (strand-I) gets re-adsorbed onto the surface.

Simulations of DNA denaturation dynamics under constrained conditions

We study the dynamics of double-stranded DNA (dsDNA) denaturation using Brownian dynamics simulations. We use a coarse-grained single nucleotide model for dsDNA which considers the helix structure. We compare the melting dynamics for free DNA of length 300 base pairs with that of a DNA of the same length but fixed from one end—mimicking DNA tethered to a substrate. We find that free DNA melts at faster rate because the entropic gain associated with denaturation is larger. Additionally, we insert the DNA in nanochannels of different widths to study the influence of the confinement on the melting dynamics. Our results suggest that there is no significant difference in the critical temperature or rate of melting when the channel width , where R g is the radius of gyration of DNA. Instead, at channel width of R g/4 we only see partial denaturation at the free DNA melting temperature. Surprisingly, this trend is reversed at higher temperature, and we find that at 110 °C tight confinement results in faster melting. This is due to the fact that confinement promotes segregation of the single-stranded segment, thereby acting as an effective entropic force aiding denaturation.

Heat induced capsid disassembly and DNA release of bacteriophage λ.

Successive structural changes of bacteriophage upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of phage melts at 87°C and its genomic DNA melts at 91°C. Interestingly, prior to the capsid melting, DNA was found to escape out of the capsid and subject to DNase digestion above 68°C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68°C, disruption of the tail first occurs and leads to the escape of DNA; above the capsid melting temperature of 87°C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.

Twist, Stretch and Melt: Quantifying How DNA Complies to Tension

Central biological processes involve continuous mechanical manipulation of DNA. In cells, DNA is constantly twisted, bended and stretched by numerous proteins mediating genome compaction, gene regulation, expression, and DNA repair. Consequently, to understand these processes, it is imperative to have an in-depth understanding of how DNA complies to mechanical stress. The helical structure and the sequence of DNA, two physical features that have a strong impact on protein-DNA interactions, are not incorporated in the current quantitative descriptions of DNA elasticity. Here we connect well-defined force-extension measurements with a novel structure-guided model for DNA elasticity, the twistable worm like chain model. This analytical description incorporates the essential physical characteristics of DNA, including how its helicity depends on extension. In addition, at forces exceeding ∼65 pN, when DNA overstretches and melts, our experimental assay exposes rich features that can be fully attributed to the underlying base sequence. An equilibrium thermodynamic model is presented that quantitatively captures this melting behaviour solely based on the knowledge of DNA sequence and elasticity. These results offer a new standard description for the mechanics of DNA and enable deeper quantitative insight into the physical interactions of DNA-associated proteins.

Quantifying how DNA stretches, melts and changes twist under tension

Understanding how DNA bends and stretches provides insight into how the genetic information it contains is expressed. However, the role that the double-helical shape of DNA plays in determining its mechanical properties has not previously been investigated. Now, a model that incorporates DNA’s famous shape provides a better understanding of how the molecule reacts to large forces.

Different pulling modes in DNA overstretching: A theoretical analysis

We study the thermally driven denaturation of a double-stranded polymer in the presence of a stretching force via Monte-Carlo simulations. When one strand only is stretched, the denaturation transition is first order, while when both strands are stretched, melting is second order. By revisiting the Poland-Scheraga model for DNA melting, we show that at room temperature, the most likely scenario is that DNA melts as it overstretches. Our results are in general agreement with the most recent experiments and suggest how varying temperature and stretching mode may help settle the question whether S-DNA exists or not.

Simplified Theory for DNA Melting Maps

DNA melting maps on DNA stretched on surfaces or in nanochannels give a coarse grained picture of the underlying sequence with potential applications in studies of structural variations and for identification of (micro)organisms.The underlying mechanism is based on the difference in free energies associated with breaking AT and GC basepairs so that DNA melts first in AT-rich regions and only at higher temperature in GC-rich regions. With a suitable choice of dye the melted regions and the unmelted regions can readily be distinguished.The Poland-Scheraga (PS)model is an Ising model with a long-range term due to the entropy associated with the single-stranded regions and, although computationally slow (∼ square of the number of basepairs), has proven to well reproduce melting data. However, by adapting our algorithms to the resolution of the experimental melting mapping (1kbp) we can make them computationally more efficient.We combine a transfer matrix approach and an exact Poland-type algorithm to study opening probabilities along DNA. We systematically explore different degrees of simplifications such as capping the long-range interactions or using a coarse-grained effective-medium approach. We evaluate our simplifications against exact solutions to the PS model for known sequences (figure).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Effects of the N-Terminal Acylamido Group of Imidazole- and Pyrrole-Containing Polyamides on DNA Sequence Specificity and Binding Affinity

The N-terminal formamido group on imidazole- and pyrrole-containing polyamides causes stacked polyamides to bind in the minor groove of DNA in the staggered motif, and it also increases the binding affinity compared to those of non-formamido compounds. To further investigate the role of the N-terminal acylamido in affecting sequence specificity and binding affinity, six polyamide analogues containing the core triheterocyclic structure IPI were designed and synthesized, and the acylamido moiety reported herein includes the following: formamido (f-IPI, 1), acetamido (Ac-IPI, 2), trifluoroacetamido (Tf-IPI, 3), N-methylureido (Mu-IPI, 4), N-methylpyrrole-2-carboxamido (PIPI, 5), and the (13)C-labeled formamido-IPI compound ((13)C-f-IPI, 6). In addition, two nonacylated IPI compounds were also synthesized and examined, namely, the amino-containing (NH(2)-IPI, 7) and non-formamido (nf-IPI, 8) compounds. The binding characteristics of compounds 1-8 were investigated using methods of molecular biology and biochemistry, which included biophysical techniques, such as DNA melts, circular dichroism, isothermal titration calorimety, and surface plasmon resonance and DNase I footprinting. With the exception of nf-IPI and NH(2)-IPI, all other compounds preferentially interacted with the cognate sequence, 5'-ACGCGT-3'. The biophysical results suggest that all six compounds bind within the minor groove at their cognate DNA sequence as stacked, staggered, antiparallel dimers. The order from highest to lowest binding affinities is as follows: f-IPI > P-IPI > Ac-IPI > Mu-IPI > Tf-IPI >> NH(2) and nf-IPI. Hence, having an acylamido moiety at the N-terminus is important for the binding of polyamides to DNA in a stacked and staggered motif. According to footprinting analysis, P-IPI (5), Ac-IPI (2), Mu-IPI (4), and Tf-IPI (3) exhibited some enhancement in sequence preference for their cognate 5'-ACGCGT-3' over f-IPI (1). NMR analysis of the [(13)C]f-IPI-CGCGnmr complex showed a slight downfield shift in the formamido (13)C signal indicating that the moiety remained intact. The trend in binding affinity suggests that steric factors play a role, in which small and planar aromatic acylamido units such as f, Ac, and P are preferred. Polar groups, such as in Mu-IPI (4) and Tf-IPI (3), afforded negative effects on binding affinity, compared to that of Ac-IPI (2).

Force induced DNA melting

When pulled along the axis, double-strand DNA undergoes a large conformational changeand elongates by roughly twice its initial contour length at a pulling force of about 70 pN.The transition to this highly overstretched form of DNA is very cooperative. Applying aforce perpendicular to the DNA axis (unzipping), double-strand DNA can also beseparated into two single-stranded DNA, this being a fundamental process inDNA replication. We study the DNA overstretching and unzipping transitionusing fully atomistic molecular dynamics (MD) simulations and argue that theconformational changes of double-strand DNA associated with either of the abovementioned processes can be viewed as force induced DNA melting. As the force atone end of the DNA is increased the DNA starts melting abruptly/smoothlyabove a critical force depending on the pulling direction. The critical forcefm, at which DNA melts completely decreases as the temperature of the system is increased.The melting force in the case of unzipping is smaller compared to the melting forcewhen the DNA is pulled along the helical axis. In the case of melting throughunzipping, the double-strand separation has jumps which correspond to the differentenergy minima arising due to sequence of different base pairs. The fraction ofWatson–Crick base pair hydrogen bond breaking as a function of force does not showsmooth and continuous behavior and consists of plateaus followed by sharp jumps.

Gold NanoStoves for Microsecond DNA Melting Analysis

In traditional DNA melting assays, the temperature of the DNA-containing solution is slowly ramped up. In contrast, we use 300 ns laser pulses to rapidly heat DNA bound gold nanoparticle aggregates. We show that double-stranded DNA melts on a microsecond time scale that leads to a disintegration of the gold nanoparticle aggregates on a millisecond time scale. A perfectly matching and a point-mutated DNA sequence can be clearly distinguished in less than one millisecond even in a 1:1 mixture of both targets.

Recovery of APOBEC3-edited human immunodeficiency virus G→A hypermutants by differential DNA denaturation PCR

Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes. As AT-rich DNA melts at lower temperatures than GC-rich DNA, use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies. The latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T(p)) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3 degrees C lower than T(p). Application of this sensitive method to two different viruses allowed us to identify human immunodeficiency virus type 1 G-->A hypermutants in a situation where none were expected and to amplify AT-rich variants selectively within a spectrum of poliovirus mutants.

Unzipping Mechanism of the Double-stranded DNA Unwinding by a Hexameric Helicase: Quantitative Analysis of the Rate of the dsDNA Unwinding, Processivity and Kinetic Step-size of the Escherichia coli DnaB Helicase Using Rapid Quench-flow Method

Kinetics of the double-stranded (ds) DNA unwinding by the Escherichia coli replicative helicase DnaB protein has been examined under single-turnover conditions using the chemical quench-flow technique. The unwinding reaction proceeds through an initial conformational transition followed by the unwinding catalytic steps and the release of the single-stranded (ss) DNA. Analyses of the reaction as a function of the number of base-pairs in the dsDNA reveal that the number of catalytic steps is not strictly proportional to the length of the dsDNA. As the helicase approaches the end of the substrate, the remaining approximately 11 bp of the DNA melts without catalytic participation of the enzyme. The kinetic step-size of the DnaB helicase, i.e. the number of the base-pairs unwound in a single catalytic step is only 1.4(+/- 0.2). The low value of the step-size indicates that the helicase unwinds a single base-pair in a single catalytic step. Thus, the DnaB helicase unzips the dsDNA in a reverse process to the zipping mechanism of the non-enzymatic double helix formation. The protein is a fast helicase that at 25 degrees C unwinds approximately 291 bp/s, much faster than previously thought, and the unwinding rate can be much higher at higher temperatures. However, the ATP-state of the enzyme has an increased dissociation rate, resulting in only a moderate unwinding processivity, P = 0.89(+/- 0.03), little dependent on the temperature. The conformational transition of the DnaB helicase-DNA complex, preceding the unwinding, is an intrinsic transition of the enzyme from the stationary conformation to the ATP-state of the helicase.

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base

The fluorescence spectroscopy of 7-azaindole ( 7aIn) incorporated in DNA oligonucleotides is investigated. Incorporation of 7aIn into DNA oligonucleotides is accomplished through standard solid-phase phosphoramidite chemistry. Fluorescence emission of the 7aIn chromophore shifts slightly to the red (from 386 nm to 388 nm) upon glycosylation at the N−1 position, but its relative fluorescence quantum yield increases 23 times, from 0.023 to 0.53. Upon incorporation into DNA, the fluorescence emission of 7aIn is greatly quenched with fluorescence quantum yields of 0.020 and 0.016 in single and double strand DNA, respectively. The fluorescence emission for 7aIn in DNA oligonucleotides shifts to the blue with an emission maximum at 379 nm. Both the strong fluorescence quenching and the blue shift of the emission spectrum signify that 7aIn is stacked with neighboring DNA bases in both single and double strand DNA. As the duplex DNA melts due to temperature increase, the fluorescence of the 7aIn chromophore increases, indicating the transition from the less fluorescent duplex DNA to the more fluorescent single strand DNA. Since this fluorescent 7aIn is a structural analog of purine, its fluorescence property may be utilized as a probe for studying nucleic acid structure and dynamics.

Mechanical Stability of Single DNA Molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In λ-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (≤10 mM NaCl), while high ionic strength buffers (1 M NaCl) stabilize the double-helical conformation. The mechanical energy that can be deposited in the DNA double helix before force induced melting occurs was found to decrease with increasing temperature. This energy correlates with the base-pairing free enthalpy Δ G bp( T) of DNA. Experiments with pure poly(dG-dC) and poly(dA-dT) DNA sequences again revealed a close correlation between the mechanical energies at which these sequences melt with base pairing free enthalpies Δ G bp(sequence): while the melting transition occurs between 65 and 200 pN in λ-phage DNA, depending on the loading rate, the melting transition is shifted to ∼300 pN for poly(dG-dC) DNA, whereas poly(dA-dT) DNA melts at a force of 35 pN.

Yeast nucleosomal particles: structural and transcriptional properties

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.5 M NaCl. Treatment of yeast nucleosomal particles with the amino group reagent dimethylmaleic anhydride is accompanied by selective release of histones H2A and H2B. The results indicate not large but significant structural differences between yeast and chicken nucleosomal cores. However, the in vitro transcription properties of complete and H2A.H2B-deficient nucleosomal cores are similar in the two kinds of particles: the histone octamer blocks RNA synthesis, this block being eliminated in part by the partial loss of histones H2A and H2B.

Evaluation of complex permittivity of aqueous solution by time domain reflectometry

Abstract A time domain reflectometry (TDR) method has been developed in order to measure dielectric relaxation process with a weak relaxation strength of the order of 0.1 in aqueous solution. Application of the TDR measurement have been made for poly(L-glutamic acid) in aqueous solution, which exhibits a helix-coil transition with changing the pH value. Dielectric relaxation process observed around 100MHz shows a definite transition in its strength in the vicinity of pH=6. The TDR method has been also applied to a DNA in aqueous solution with 0.1SSC buffer. A double helix structure of DNA melts at about 75°C to a coiled structure. Relaxation process around 100MHz shows a transition in the strength and also in the relaxation time around this temperature. In both cases, relaxation process caused by water molecules could be observed separately from the process observed around 100MHz. The relaxation strength and the relaxation time are nearly the same as those of the free water. A bilinear analysis developed by Cole has been used to measure methanol-water mixtures. A relaxation process could be observed continuously with the composition. It has been concluded that the bilinear analysis is quite powerful if the present TDR method is used together for the dielectric measurement covering a wide frequency region from 1MHz to 15GHz.

Qualitative changes in DNA indicating differential DNA replication during early embryogenesis of the newt Triturus vulgaris

During gastrulation of the newt Triturus vulgaris considerable changes in the melting behaviour and in the CsCl density gradient pattern of DNA occur. The melting curves of DNAs from mid to late gastrulae (stages 11b-12c) deviate from the regular shape. Whereas the Tm values are identical in the stages 11a (early gastrula) and 15 (early neurula), and correspond to the standard DNA (stage 36 = tailbud), a significant rise of Tm (0.8-1.2 degrees C) has been recorded in the stages 12a/b (yolk plug). The differences in melting behaviour become visible by the deviation of the curves above Tm. These deviations from the normal sigmoidal shape are caused by the fact that a portion of DNA melts at higher temperatures than usual. Therefore the thermal denaturation of DNA is completed at approximately 3-4 degrees C later than in standard DNA. Both the derivative curves and the plots on normal probability paper demonstrate a heterogeneity of DNA in the stages 11b-12c which indicates the presence of an additional GC-rich satellite fraction. These findings are confirmed by CsCl density gradient studies. Thus, in the stages 12a/b a slight shoulder on the heavy side of the gradients occurs, being absent in the other stages. From these facts we have to conclude that there is a stage-dependent multiplication and elimination of GC-rich sequences.

The structure of kinetoplast DNA: I. Properties of the intact multi-circular complex from Crithidia luciliae

1. We have developed a modified isolation procedure that yields kinetoplast DNA networks containing more than 90% closed circular DNA, as judged by two criteria: 1. (a) In 0.15 M NaCl 0.015 M sodium citrate (pH 7.0), less than 10% of the intact kinetoplast DNA melts in the temperature region of sonicated kinetoplast DNA. In 7.2 M NaClO 4 the kinetoplast DNA melts with a T m 26° C higher than sonicated kinetoplast DNA. Even after complete melting in 7.2 M NaClO 4 at 90° C, the network remains intact, as judged by regain of hypochromicity on cooling and analysis in CsCl containing propidium diiodide. 2. (b) In alkaline sucrose gradients more than 90% of the kinetoplast DNA sediments in a single peak. 2. In CsCl gradients containing ethidium bromide or propidium diiodide intact kinetoplast DNA gives a single uni-modal band showing an extremely restricted dye uptake. From the position of the band relative to the bands of PM2 DNA, the superhelix density of these networks is calculated to be +3.9 twists per 1000 base pairs. The superhelix density of closed mini-circles, efficiently liberated from the networks by shear in a French press, is −0.5 twists per 1000 base pairs. We attribute the high superhelix density (the highest yet observed in any DNA) of intact networks to their compact, highly catenated structure, leading to an additional constraint on dye uptake, superimposed on the restriction due to closed circularity.

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm(3) has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 microm; it reassociates with a low C(0)t(1/2) after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm(3). MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm(3), slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.

Partial purification of native rRNA and tRNA cistrons from mycoplasma sp. (Kid).

Precise optical melting profiles of purified DNA from Mycoplasma sp. (Kid) show a secondary hyperchromic rise, corresponding to 1.4 per cent of the total DNA, occurring at 88 degrees C, while the bulk of the DNA melts at 79.5 degrees C, indicating an average base composition of 24.9 per cent guanine-cytosine (G-C). A method is presented, using sonication followed by hydroxyapatite column chromatography, for the partial purification of regions of a genome which contain significantly higher G-C than the average value for the genome. The procedure does not involve denaturation and renaturation of the high G-C material so that purified DNA is in its native, double-stranded state and has a normal melting profile. When applied to Mycoplasma sp. (Kid), the method yielded a fraction of native DNA enriched 40 times with respect to those regions coding for rRNA and tRNA. This enriched DNA has a saturation hybridization value of 15.9 per cent with Kid rRNA plus tRNA. The saturation hybridization values of the bulk DNA with rRNA and tRNA are 0.26 per cent and 0.16 per cent, respectively. Based on a genome size of 6.84 x 10(8), obtained by electron microscopy, this indicates that Mycoplasma sp. (Kid) contains only enough ribosomal DNA to code for one set of 23S plus 16S rRNA and only enough DNA complementary to tRNA to code for 44 different tRNA molecules.