DNA Molecules In Solution Research Articles

Reentrant Behavior of Divalent-Counterion-Mediated DNA-DNA Electrostatic Interaction

The problem of DNA-DNA interaction mediated by divalent counterions is studied using computer simulation. Although divalent counterions cannot condense free DNA molecules in solution, we show that if DNA configurational entropy is restricted, divalent counterions can cause DNA reentrant condensation similar to that caused by tri- or tetravalent counterions. DNA-DNA interaction is strongly repulsive at small or large counterion concentration and is negligible or slightly attractive for a concentration in between. Implications of our results to experiments of DNA ejection from bacteriophages are discussed. The quantitative result serves to understand electrostatic effects in other experiments involving DNA and divalent counterions.

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA

It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH(3))(2)}(2)(micro-OH)(micro-1,2,3-ta-N (1),N (2))](NO(3))(2) (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient

Solid-state nanopores are sensors capable of analyzing individual unlabelled DNA molecules in solution. While the critical information obtained from nanopores (e.g., DNA sequence) is the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here we study the process of DNA capture into nanofabricated silicon nitride pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 basepairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. In addition, we show that application of a 20-fold salt gradient enables detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analyzing unamplified DNA samples using nanopores.

Electrical characterization of DNA molecules in solution using impedance measurements

We report on impedance measurements of fluids to examine the impact of the length and concentration of free-floating double-stranded DNA molecules. The impedance versus frequency characteristics were fitted to an equivalent circuit model including solution dielectric capacitance and conductance. The extraction of these parameters can be used to detect the presence of DNA molecules in the nanomolar range for a 400bp long molecule. Our results show that the extracted dielectric capacitance and conductance increase with molecule length and concentration owing to a corresponding increase in number of molecule dipoles and counterions in solution.

Synthesis of cobalt ferrite core/metallic shell nanoparticles for the development of a specific PNA/DNA biosensor

Controlled synthesis of cobalt ferrite superparamagnetic nanoparticles covered with a gold shell has been achieved by an affinity and trap strategy. Magnetic nanoparticles are functionalized with a mixture of amino and thiol groups that facilitate the electrostatic attraction and further chemisorption of gold nanoparticles, respectively. Using these nanoparticles as seeds, a complete coating shell is achieved by gold salt-iterative reduction leading to monodisperse water-soluble gold-covered magnetic nanoparticles, with an average diameter ranging from 21 to 29 nm. These constitute a versatile platform for immobilization of biomolecules via thiol chemistry, which is exemplified by the immobilization of peptide nucleic acid (PNA) oligomers that specifically hybridize with complementary DNA molecules in solution. Hybridation with DNA probes has been measured using Rhodamine 6G fluorescence marker and the detection of a single nucleotide mutation has been achieved. These results suggest the PNA-nanoparticles application as a biosensor for DNA genotyping avoiding commonly time-consuming procedures employed.

Surface-enhanced Raman scattering of λ -DNA

By using the molecular combing method, we stretched DNA molecules with colloidal silver nanoparticles adsorbed on them. Surface-enhanced Raman scattering (SERS) spectra were recorded in this particular DNA/Ag system. We also studied SERS spectra of DNA molecules in solution. The spectra of DNA in different concentration solution show distinct differences, and we speculate that the coiling states of DNA molecules are different in different concentrations, leading to the spectral differences.

On Bifurcations of Equilibria of Intrinsically Curved, Electrically Charged, Rod-like Structures that Model DNA Molecules in Solution

DNA molecules in the familiar Watson–Crick double helical B form can be treated as though they have rod-like structures obtained by stacking dominoes one on top of another with each rotated by approximately one-tenth of a full turn with respect to its immediate predecessor in the stack. These “dominoes” are called base pairs. A recently developed theory of sequence-dependent DNA elasticity (Coleman, Olson, & Swigon, J. Chem. Phys. 118:7127–7140, 2003) takes into account the observation that the step from one base pair to the next can be one of several distinct types, each having its own mechanical properties that depend on the nucleotide composition of the step. In the present paper, which is based on that theory, emphasis is placed on the fact that, as each base in a base pair is attached to the sugar-phosphate backbone chain of one of the two DNA strands that have come together to form the Watson–Crick structure, and each phosphate group in a backbone chain bears one electronic charge, two such charges are associated with each base pair, which implies that each base pair is subject to not only the elastic forces and moments exerted on it by its neighboring base pairs but also to long range electrostatic forces that, because they are only partially screened out by positively charged counter ions, can render the molecule’s equilibrium configurations sensitive to changes in the concentration c of salt in the medium. When these electrostatic forces are taken into account, the equations of mechanical equilibrium for a DNA molecule with N + 1 base pairs are a system of μN non-linear equations, where μ, the number of kinematical variables describing the relative displacement and orientation of adjacent pairs is in general 6; it reduces to 3 when base-pair steps are assumed to be inextensible and non-shearable. As a consequence of the long-range electrostatic interactions of base pairs, the μN × μN Jacobian matrix of the equations of equilibrium is full. An efficient numerically stable computational scheme is here presented for solving those equations and determining the mechanical stability of the calculated equilibrium configurations. That scheme is employed to compute and analyze bifurcation diagrams in which c is the bifurcation parameter and to show that, for an intrinsically curved molecule, small changes in c can have a strong effect on stable equilibrium configurations. Cases are presented in which several stable configurations occur at a single value of c.

Effect of Water Drag on Diffusion of Drifting Polarons in DNA

It has been shown, theoretically and experimentally, that a hole or an excess electron on a DNA molecule in solution forms a delocalized wave function, a polaron. For an all-adenine (A) sequence or a mixed sequence of guanines (G's) and A's, calculations taking into account the polarization of the solution give the wave function spread over approximately four bases, which appears to be in agreement with experiment. The polaron may move by hopping or by drift. Drift can take place in a region with all the same bases, for example, A's, by the polaron dropping an A on the trailing edge and picking up an A on the leading edge. For drift that is not too rapid, the necessity of the polarization changing as the polaron moves exerts a drag on the polaron. We calculate the drag by using a model introduced earlier to describe the polaron. We find the drag to be proportional to the velocity of the polaron and to the orientational relaxation time of the water molecules. The drag is also a function of the Coulomb interactions of the fractional charges on the bases constituting the polaron, as modified by the polarization charge induced in the solution. The diffusion rate and mobility for all A polarons, calculated taking into account the drag, are 8 x 10(-5) cm(2)/s and 3 x 10(-3) cm(2)/(V s), respectively. We believe that in the experimental studies that have been carried out on hole propagation in a series of A's it was drift being observed rather than the hopping of a localized hole between adjacent A's, as was assumed to be the case.

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.

Helicoidal transfer matrix model for inhomogeneous DNA melting

An inhomogeneous helicoidal nearest-neighbor model with continuous degrees of freedom is shown to predict the same DNA melting properties as traditional long-range Ising models, for free DNA molecules in solution, as well as superhelically stressed DNA with a fixed linking number constraint. Without loss of accuracy, the continuous degrees of freedom can be discretized using a minimal number of discretization points, yielding an effective transfer matrix model of modest dimension (d=36). The resulting algorithms to compute DNA melting profiles are both simple and efficient.

Conformational changes of DNA molecules in interactions with bioactive compounds. I. Influence of metal ions on the conformation of DNA molecules in solution

The influence of metal ions of different valence on the conformation of DNA molecule in solution has been studied. The influence of concentration and charge of counterions on the volume, persistent length and secondary structure of the macromolecules was analyzed. An assessment of the permeability of DNA coil for the solvent at different values of ionic strength of the solution was made. The state of the DNA molecule in solutions with high ionic strengths, when the presence of certain ions causes sharp changes in optical anisotropy of the macromolecule, is considered in detail.

DARTs: A DNA-based in vitro polypeptide display technology.

Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.

Equilibrium adsorption of ligands on DNA (for chitosan)

The binding of ligands to DNA molecules in solution is characterized by a distribution function determining the probability that a certain number of ligands per DNA molecule are adsorbed. As a result of the binding to polycations, rigid linear double-stranded DNA molecules (with negative charges of the phosphate groups neutralized by the positive charges of ligands) exhibit a phase transition. Experimental data on the binding of chitosan to DNA are interpreted assuming that chitosan exhibits equilibrium adsorption on DNA. When the number of chitosan molecules adsorbed on DNA exceeds a certain critical value, the DNA molecule covered by chitosan becomes capable of interacting with like DNA molecules. This interaction (attraction) results in the formation of particles constituting a liquid-crystalline dispersion. It is shown that the proposed model with certain parameters provides a description of certain experimental data characterizing the formation of cholesteric liquid-crystalline dispersions. An analysis of the experimental data makes it possible to determine both the size of a site occupied by an amino sugar unit of chitosan adsorbed on DNA and the energy of interaction of this unit with DNA.

Controlled Assembly of Monolayer-Protected Gold Clusters by Dissolved DNA

One-dimensional chains of gold nanoparticles coated with cationic trimethyl(mercaptoundecyl)ammonium (TMA) monolayers have been electrostatically assembled along DNA molecules in solution by careful control of the relative molar quantities of nanoparticles and DNA base pairs. The nanoparticles are monolayer-protected Au clusters (MPCs) with average diameter 4.4 nm. Transmission electron microscopy (TEM) images of dried solution droplets in which the DNA base pair/MPC mole ratio was about 20:1 display chains of nanoparticles a few microns long in which the Au cores are lined up with nearly contacting cationic monolayer edges along the anionic DNA template. The gold core edge-edge distance in the TEM image is 2.9±0.9 nm, which is roughly twice the TMA monolayer thickness. The linear structures formed at a 10:1 DNA base pair/MPC mole ratio appear to persist stably in solutions. The Au surface plasmon resonance band in UV−vis spectra intensifies and red-shifts as the DNA/MPC mole ratio is increased; precipita...

Trapping of DNA by dielectrophoresis.

Under suitable conditions, a DNA molecule in solution will develop a strong electric dipole moment. This induced dipole allows the molecule to be manipulated with field gradients, in a phenomenon known as dielectrophoresis (DEP). Pure dielectrophoretic motion of DNA requires alternate current (AC) electric fields to suppress the electrophoretic effect of the molecules net charge. In this paper, we present two methods for measuring the efficiency of DEP for trapping DNA molecules as well as a set of quantitative measurements of the effects of strand length, buffer composition, and frequency of the applied electric field. A simple configuration of electrodes in combination with a microfluidic flow chamber is shown to increase the concentration of DNA in solution by at least 60-fold. These results should prove useful in designing practical microfluidic devices employing this phenomenon either for separation or concentration of DNA.

Orientation and Positioning of DNA Molecules with an Electric Field Technique

A controlled handling of single molecules is essential for the fabrication and the investigation of devices based on molecules. We present here the implementation of an electric field based method used to manipulate DNA molecules by means of lithographically patterned metallic electrodes. We optimized the geometry of the lithographic structures to favor a precise positioning of the molecules via dielectrophoresis. This process is combined with an orientation of the molecules parallel to the electric field lines due to their induced dipole moment. The relatively high polarizability of the DNA molecules in solution is essential to achieve these manipulations. We expect this method to be softer than the stretching of molecules using a receding meniscus. The visualization of the molecules was achieved using fluorescence microscopy.

Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array.

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3' and 5' overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

HIV-1 Preintegration Complexes Preferentially Integrate into Longer Target DNA Molecules in Solution as Detected by a Sensitive, Polymerase Chain Reaction-based Integration Assay

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

Dynamics of dilute and semidilute DNA solutions in the start-up of shear flow

We have investigated the dynamics of dilute (10−5C*) and semidilute (⩽6C*) DNA solutions both in steady and in the start-up of shear flow by combining fluorescence microscopy, bulk rheological measurements, and Brownian dynamics simulations. First, the microscopic states, i.e., the conformational dynamics of single DNA molecules in solution during the start-up of shear flow, were examined by fluorescence microscopy. To investigate the macroscopic response resulting from the changes in the microscopic state, the bulk shear viscosity of the same DNA solutions was also measured. While the transient dynamics of individual molecules is highly variable, an overshoot in the ensemble-averaged molecular extension is observed above a critical Wi following an overshoot in shear viscosity for both dilute and semidilute DNA solutions. These two overshoots are further analyzed and explained on a physical basis from our simulation findings. Based on the physical picture, we have derived a simple scaling to predict the s...