Probability distribution analysis of force induced unzipping of DNA

We present a semimicroscopic model of dsDNA by incorporating the directional nature of hydrogen bond to describe the force induced unzipping transition. Using exact enumeration technique, we obtain the force-temperature and the force-extension curves and compare our results with the other models of dsDNA. The model proposed by us is rich enough to describe the basic mechanism of dsDNA unzipping and predicts the existence of an "eye phase." We show oscillations in the probability distribution function during unzipping. Effects of stacking energies on the melting profile have also been studied.     

Dynamic Release of Bending Stress in Short dsDNA by Formation of a Kink and Forks

Bending with high curvature is one of the major mechanical properties of double‐stranded DNA (dsDNA) that is essential for its biological functions. The emergence of a kink arising from local melting in the middle of dsDNA has been suggested as a mechanism of releasing the energy cost of bending. Herein, we report that strong bending induces two types of short dsDNA deformations, induced by two types of local melting, namely, a kink in the middle and forks at the ends, which we demonstrate using D‐shaped DNA nanostructures. The two types of deformed dsDNA structures dynamically interconvert on a millisecond timescale. The transition from a fork to a kink is dominated by entropic contribution (anti‐Arrhenius behavior), while the transition from a kink to a fork is dominated by enthalpic contributions. The presence of mismatches in dsDNA accelerates kink formation, and the transition from a kink to a fork is removed when the mismatch size is three base pairs.     

Probing the mechanical stability of DNA in the presence of monovalent cations

We examine the interaction between monovalent cations and DNA using several different assays that measure the stability of double-stranded DNA (dsDNA). The thermal melting of dsDNA and the mechanical separation of dsDNA into two single strands both depend on the stability of dsDNA with respect to ssDNA and are sensitive to the interstrand phosphate repulsion. We find that the experimentally measured melting temperatures and unzipping forces are approximately the same for all of the ions considered in this study. Likewise, the force required to transform B-DNA into the overstretched form is also similar for all of the ions. In contrast, for a given ion concentration, the force at which the overstretched state fully relaxes back to the canonical B-DNA form depends on the cation; however, for all cations, the overstretching force decreases with decreasing ion concentration, suggesting that this force is sensitive to screening. We observe a general trend for smaller ions to produce more efficient relaxation. Finally, for a given cation, the relaxation can also depend on the anion.     

The probability analysis of opening of DNA

We have studied the separation of a double stranded DNA (dsDNA), which is driven by either the temperature or force. By monitoring the probability of opening of entire base pairs along the chain, we show that the opening of a dsDNA depends not only on the sequence but also on the constraints on the chain in the experimental setups. Our results clearly demonstrate that the force-induced melting of dsDNA, whose one of the ends is constrained, is significantly different from the thermal melting, when both ends are free.     

Adsorption and hybridization of oligonucleotides on mercaptoacetic acid-capped CdSe/ZnS quantum dots and quantum dot-oligonucleotide conjugates

Interest in the unique optical properties of quantum dots (QDs) has resulted in the development QD-bioconjugates for imaging and diagnostics. Although these applications are numerous, considerably less is known about the interactions between QDs and biomolecules. In this work, we describe hydrogen-bonding interactions between oligonucleotides and CdSe/ZnS quantum dots capped with mercaptoacetic acid ligands. The strength of the interactions can be modulated by changes in the pH and ionic strength, the addition of formamide, and differences between ssDNA and dsDNA. Fluorescence resonance energy transfer experiments have shown that conjugated oligonucleotides adopt a conformation that lies across the surface of the QD. The hydrogen-bonding interactions also affect the kinetics of hybridization with QD-DNA conjugates and the thermal stability of QD-conjugated dsDNA. The former is analogous to conventional solid-phase hybridization, where stronger oligonucleotide adsorption leads to faster kinetics. With respect to the latter, interactions with the QD surface can sharpen the melt transition and alter the melt temperature of dsDNA. These effects are largely absent when adsorptive interactions are minimized.     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Minor groove binding of a novel tetracationic diviologen

Novel tetracationic diviologen compounds of the general formula CH3(CH2)nV2+(CH2)6V2+(CH2)nCH3 (where V2+ = 4,4'-bipyridinium and n = 5 or 11) were investigated as electrochemical reporters of DNA duplex formation. These compounds bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) when the DNA is either present in solution or immobilized at electrode surfaces. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for the reduction of the V2+ state to the V+ (viologen radical cation) state. An analysis of the charge for this reduction provided isotherms and binding constants for binding of these diviologens to both forms of immobilized DNA. Saturation of the binding is achieved at solution concentrations near 20 microM. For both the n = 5 and 11 diviologens, binding to ssDNA is driven by electrostatic charge neutralization. For the n = 11 case, the binding is cooperative. In the presence of dsDNA, the n = 11 diviologen exhibits a unique reduction potential for the V2+/+ redox couple that is shifted approximately 100 mV negative of that in the presence of ssDNA. This new electrochemical signature is attributed to the reduction of viologen groups bound in the minor groove of the DNA duplex. For dsDNA in solution, an increase in the thermal denaturation temperature (Tm) from 60 to 66 degrees C as a function of the n = 11 diviologen concentration confirmed its interaction with the duplex. Circular dichroism (CD) spectroscopy also was used to investigate the binding of both the V2+ and V+ redox states of the n = 11 diviologen to dsDNA in solution. For the V+ state, a CD signal was observed that is consistent with the presence of face-to-face pi dimers of the viologen groups. This unambiguously demonstrates the binding of this redox state of the diviologen in the dsDNA minor groove and the formation of such dimers in the minor groove.     

Thermoresponsive polymeric gel as a medium for examining interactions between dsDNA and an anticancer drug

A piece of dry N-isopropylacrylamide polymer was soaked in phosphate buffer to obtain a hydrogel which was then employed in the examination of interactions between an anticancer drug C-1311 (5-diethylaminoethyl-amino-8-hydroxyimidazoacridinone) and dsDNA. dsDNA was introduced into the polymer at the polymerization stage. The drug was added to the buffer. Using the volume phase transition of the gel at 40 °C, the unbound drug could be determined in the solution released during the transition, which made the calculations more reliable. The interaction parameters were calculated using the McGhee and von Hippel model. It appeared that in the gel medium, the interaction between the drug and dsDNA is spatially limited, since the number of binding units of the polymer chain occupied by one drug molecule was found to be one, while it was two in the regular buffer solution. Figure   The two authors Agata Kowalczyk and Anna M. Nowicka contributed equally to this work.     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.     

Selective enzymatic labeling to detect packing-induced denaturation of double-stranded DNA at interfaces

The adsorption of DNA on surfaces is a widespread procedure and is a common way for fabrication of biosensors, DNA chips, and nanoelectronic devices. Although the biologically relevant and prevailing in vivo structure of DNA is its double-stranded (dsDNA) conformation, the characterization of DNA on surfaces has mainly focused on single-stranded DNA (ssDNA). Studying the structure of dsDNA on surfaces is of invaluable importance to microarray performance since their effectiveness relies on the ability of two DNA molecules to hybridize and remain stable. In addition, many of the enzymatic transactions performed on DNA require dsDNA, rather than ssDNA, as a substrate. However, it is not established that adsorbed dsDNA remains in its structure and does not denature. Here, two methodologies have been developed for distinguishing between surface-adsorbed single- and double-stranded DNA. We demonstrate that, upon formation of a dense monolayer, the nonthiolated strand comprising the dsDNA is released and the monolayer consists of mostly ssDNA. The fraction of dsDNA within the ssDNA monolayer depends on the length of the oligomers. A likely mechanism leading to this rearrangement is discussed.     

Shot-noise limited single-molecule FRET histograms: comparison between theory and experiments

We describe a simple approach and present a straightforward numerical algorithm to compute the best fit shot-noise limited proximity ratio histogram (PRH) in single-molecule fluorescence resonant energy transfer diffusion experiments. The key ingredient is the use of the experimental burst size distribution, as obtained after burst search through the photon data streams. We show how the use of an alternated laser excitation scheme and a correspondingly optimized burst search algorithm eliminates several potential artifacts affecting the calculation of the best fit shot-noise limited PRH. This algorithm is tested extensively on simulations and simple experimental systems. We find that dsDNA data exhibit a wider PRH than expected from shot noise only and hypothetically account for it by assuming a small Gaussian distribution of distances with an average standard deviation of 1.6 A. Finally, we briefly mention the results of a future publication and illustrate them with a simple two-state model system (DNA hairpin), for which the kinetic transition rates between the open and closed conformations are extracted.     

Direct energy transfer from conjugated polymer to DNA intercalated dye: label-free fluorescent DNA detection

The development of methods for DNA detection is of importance in disease diagnosis, gene-targeted drug discovery and molecular biology field. In this paper, we synthesize a new cationic water-soluble CP containing fluorene moiety and flexible ethylenic moiety in the backbone (PFV) for label-free DNA detection. The conformational freedom of PFV provides stronger interactions with double-stranded DNA (dsDNA) and optimizes the orientation of transition moments between PFV and ethidium bromide (EB) intercalated in dsDNA. The efficient FRET from PFV (donor) to EB (acceptor) intercalated in dsDNA is observed and the emission of EB is amplified by the good light-harvesting ability of conjugated polymers. The interactions between PFV and DNA can also be probed by measuring the FRET ratio between PFV and EB intercalated in DNA. In comparison to other DNA detection assays based on FRET and conjugated polymers, synthesis of dye-labeled DNA probe is avoided in our method, which significantly reduces the cost and the synthetic complexity. The PFV/dsDNA/EB system provides promising applications on DNA detection with a simply, fast and label-free manner.     

Spectroscopic Electrochemical Studies of Interaction Between Fuchsin Basic DNA

Visible spectroscopic and electrochemical methods were used to study the interactions between DNA and fuchsin basic(FB). FB has an irreversible electro-oxidation peak in 5 mmol/L Tris-HCl buffer solution at pH = 7.4 on a glassy carbon electrode(GCE). After adding certain concentration of dsDNA, the oxidation peak current of FB decreases, but the peak potential hardly changs. The visible absorption spectroscopic study shows that the binding mode of FB to dsDNA is intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is smaller than 0. 2, and a new substance, which produces a new absorption peak, is obtained via a covalent binding between dsDNA and FB apart from intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is larger than 0. 2. The visible absorption spectra varies no longer when the ratio of the concentration of dsDNA to FB is larger than 1.5. A mean binding ratio of dsDNA to FB was determined to be 1.4: 1,suggesting that two complexes FB-dsDNA and FB-2dsDNA be formed. The interaction between FB and ssDNA was only electrostatic binding. The more powerful interaction of FB with dsDNA than with ssDNA may be applied for the recognition of dsDNA and ssDNA, and in DNA biosensor as hybridization indicator.     

Cytosine methylation regulates DNA bendability depending on the curvature

Cytosine methylation plays an essential role in many biological processes, such as nucleosome inactivation and regulation of gene expression. The modulation of DNA mechanics may be one of the regulatory mechanisms influenced by cytosine methylation. However, it remains unclear how methylation influences DNA mechanics. Here, we show that methylation has contrasting effects on the bending property of dsDNA depending on DNA curvature. We directly applied bending force on 30 base pairs of dsDNA using a D-shaped DNA nanostructure and measured the degree of bending using single-molecule fluorescence resonance energy transfer without surface immobilization. When dsDNA is weakly bent, methylation increases the stiffness of dsDNA. The stiffness of dsDNA increased by approximately 8% with a single methylation site for 30 bp dsDNA. When dsDNA is highly bent by a strong force, it forms a kink, i.e., a sharp bending of dsDNA. Under strong bending, methylation destabilizes the non-kink form compared with the kink form, which makes dsDNA near the kink region apparently more bendable. However, if the kink region is methylated, the kink form is destabilized, and dsDNA becomes stiffer. As a result, methylation increases the stiffness of weakly bent dsDNA and concurrently can promote kink formation, which may stabilize the nucleosome structure. Our results provide new insight into the effect of methylation, showing that cytosine methylation has opposite effects on DNA mechanics depending on its curvature and methylation location.

D-shaped DNA is used to observe dsDNA bending mechanics. Cytosine methylation increases the intrinsic stiffness of dsDNA. Under strong bending, methylation stabilizes or destabilizes a kink form depending on methylation sites.     

Electrochemical Characteristics of a Novel Pyridinium Salt as a Candidate Drug Molecule and Its Interaction with DNA

In this article, for the first time, the electrochemical properties of a novel pyridine derivative, 4‐(2‐(2‐hydroxybenzylidene) hydrazinyl)‐1‐(3‐phenylpropyl) pyridinium bromide (abbreviated as 4‐Pyri), and its interaction with double stranded DNA (dsDNA) was investigated. The interaction between candidate drug molecule (4‐Pyri) and dsDNA was analyzed by examining 4‐Pyri (+0.6 V and +0.8 V) and guanine (+1.0 V) oxidation signal changes with Differential Pulse Voltammetry (DPV) and Cyclic Voltammetry (CV). Electrochemical Impedance Spectroscopy (EIS) was used to show the resistance changes before and after the interaction between 4‐Pyri and dsDNA. We showed that after the interaction with 4‐Pyri, the oxidation currents of guanine decreased dramatically, whereas the intrinsic oxidation currents of 4‐Pyri dramatically increased. 4‐Pyri oxidation current differences before and after the interaction with dsDNA enabled us to determine such interaction separately from guanine oxidation signals. In addition, resistance differences were observed at before and after the interaction with each other that confirmed the possible interaction. In addition, toxicity effect (S%) value, which is an important parameter for electrochemical studies indicated 4‐Pyri's toxicity to dsDNA. Our results demonstrated that 4‐Pyri interacts with dsDNA, and could be used as a potential candidate drug molecule due to its remarkable impact on dsDNA.     

Al3+/graphene composites for electrochemical detection of DNA cleavage

Herein, we present the electrochemical co-deposition of Al3+/graphene composites directly from an aqueous mixture containing graphene oxide (GO) and Al3+. The obtained Al3+/graphene composites with good electrochemical activity were regarded as an appropriate immobilization platform for double-stranded DNA (dsDNA). The nontoxic redox probe xanthurenic acid (XA) was successfully applied to recognize single-stranded DNA and dsDNA. We illustrated that the scission of dsDNA caused by GO combining with some metal ions could be detected by monitoring the electrochemical signals of XA.     

Influence of percentage of guanine molecules, OH radicals, UV irradiation and temperature on electrooxidation of short synthetic oligonucleotides

The electrooxidation of short synthetic 20-nucleotides DNA sequences with various amount of guanine molecules has been studied in a wide temperature range by square wave voltammetry and the results were compared with UV-vis and CD spectra. A twofold increase of dsDNA voltammetric peak, related to an increase in the number of electrons transferred in the guanine electrooxidation process was found to begin at a temperature lower by circa 20 °C compared to the well known increase of the dsDNA absorbance upon denaturation. Since the dsDNA voltammetric peaks are related directly to the electrooxidation of guanine and adenine, early conformational changes in dsDNA are responsible for this effect. An increase in percentage of guanine in the DNA chains caused a delay in the conformational, predenaturation changes. An exception to this behavior was found for polyguanine (100% guanine). Interestingly, two distinct ranges of change in ellipticity in the CD spectra correlate well with the changes obtained by voltammetry. We have also checked the influence of OH radicals and UV irradiation on the dsDNA oxidation.     

Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay

We demonstrate that the gel electrophoretic mobility-shift assay (EMSA) can be used for site-selective and quantitative monitoring of nicks in linear double-stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well-detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence-specifically generated by a nick. Since nick-induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest-neighbor energetics.     

Additional base-pair formation in DNA duplexes by a double-headed nucleotide

We have designed and synthesised a double-headed nucleotide that presents two nucleobases in the interior of a dsDNA duplex. This nucleotide recognises and forms Watson-Crick base pairs with two complementary adenosines in a Watson-Crick framework. Furthermore, with judicious positioning in complementary strands, the nucleotide recognises itself through the formation of a T:T base pair. Thus, two novel nucleic acid motifs can be defined by using our double-headed nucleotide. Both motifs were characterised by UV melting experiments, CD and NMR spectroscopy and molecular dynamics simulations. Both motifs leave the thermostability of the native dsDNA duplex largely unaltered. Molecular dynamics calculations showed that the double-headed nucleotides are accommodated in the dsDNA by entirely local perturbations and that the modified duplexes retain an overall B-type geometry with the dsDNA unwound by around 25 or 60°, respectively, in each of the modified motifs. Both motifs can be accommodated twice in a dsDNA duplex without incurring any loss of stability and extrapolating from this observation and the results of modelling, it is conceivable that both can be multiplied several times within a dsDNA duplex. These new motifs extend the DNA recognition repertoire and may form the basis for a complete series of double-headed nucleotides based on all 16 base combinations of the four natural nucleobases. In addition, both motifs can be used in the design of nanoscale DNA structures in which a specific duplex twist is required.