Spectroscopic Characterization of the Interaction of Phenosafranin and Safranin O with Double Stranded, Heat Denatured and Single Stranded Calf Thymus DNA

Interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA has been studied by fluorescence, absorbance and circular dichroic techniques. Binding to the double stranded and heat denatured DNA conformations induced strong quenching in the fluorescence spectra of both dyes. Linear Scatchard plots indicated the binding to be of one type and the affinity evaluated to be of the order of 10(5) M(-1) with double stranded and heat denatured DNAs. Fluorescence quenching was much weaker with the single stranded DNA and the binding affinity was one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of the dye molecules bound to the double stranded and heat denatured DNAs was quenched much less compared to that bound to the single stranded DNA. Further, there was significant emission polarization for the bound dyes and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding. Salt dependence of the binding phenomenon revealed that electrostatic forces have significant role in the binding process. The intercalation of these molecules to double stranded and heat denatured DNA and simple stacking to single strands was proved by these fluorescence techniques. Support to the fluorescence results have been derived from absorption and circular dichroic results. Phenosafranin was revealed to be a stronger binding species compared to safranin O.     

Thionine Interaction to DNA: Comparative Spectroscopic Studies on Double Stranded Versus Single Stranded DNA

Interaction of thionine with double stranded and single stranded calf thymus DNA has been studied by absorbance, fluorescence, competition dialysis, circular dichroism and isothermal titration calorimetry. Binding to the native double stranded DNA conformation induced strong quenching in fluorescence spectrum of thionine. Linear Scatchard plots indicated the binding to be of one type and the affinity values evaluated to be of the order of 10⁵ M⁻¹ with double stranded DNA. Fluorescence quenching was much weaker with single stranded DNA and the binding affinity was about one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of dye molecules bound to the double stranded DNA was quenched much less compared to those bound to the single stranded DNA. Furthermore, there was significant emission polarization for the bound dye molecules and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding to ds DNA. Salt dependence of the binding phenomenon revealed that electrostatic forces played a significant role in the binding process. The intercalation of the dye molecules to double stranded DNA and simple stacking to single strands was proved from these fluorescence techniques. Support to the fluorescence results have been derived from absorption, circular dichroic and dialysis results. Calorimetric studies suggested that the binding to ds DNA conformation was both enthalpy and entropy favoured while that to ss DNA was predominantly entropy favoured.     

Influence of the sequence on the ab initio band structures of single and double stranded DNA models

The solid state physical approach is widely used for the characterization of electronic properties of DNA. In the simplest case the helical symmetry is explicitly utilized with a repeat unit containing only a single nucleotide or nucleotide pair. This model provides a band structure that is easily interpretable and reflects the main characteristic features of the single nucleotide or a nucleotide pair chain, respectively. The chemical variability of the different DNA chains is, however, almost completely neglected in this way. In the present work we have investigated the effect of the different sequences on the band structure of periodic DNA models. For this purpose we have applied the Hartree–Fock crystal orbital method for single and double stranded DNA chains with two different subsequent nucleotides in the repeat unit of former and two different nucleotide pairs in the latter case, respectively. These results are compared to simple helical models with uniform sequences. The valence and conduction bands related to the stacked nucleotide bases of single stranded DNA built up only from guanidine as well as of double stranded DNA built up only from guanidine–cytidine pairs showed special properties different from the other cases. Namely, they had higher conduction and lower valence band positions and this way larger band gaps and smaller widths of these bands. With the introduction of non-uniform guanidine containing sequences band structures became more similar to each other and to the band structures of other sequences without guanidine. The maximal bandwidths of the non-uniform sequences are considerably smaller than in the case of uniform sequences implying smaller charge carrier mobilities both in the conduction and valence bands.     

Scanning tunneling spectrosopy study of DNA conductivity

We used STM to study the conductivity of 32 nucleotide long DNA molecules chemically attached to a gold surface. Two oligonucleotides containing all four base types namely G, A, C, T, one single stranded and one double helical, all showed conductance data significantly higher than DNA containing only T and A that were either single stranded d(T32) or double helical d(T32).d(A32) in confirmation. Within each sequence group, the conductivity of the double helical form was always higher than that of the single strand. We discuss the impact of structure, particular base stacking and affinity to the phase transition.      

Biomolecular self-assembly of micrometer sized silica beads on patterned glass substrates

A self-assembly process for the two-dimensional arrangement of micrometer sized silica beads on glass slides was developed. It is based on the hybridization of two single stranded DNA-oligonucleotides to a DNA double helix. To prepare for the self-assembly process the silica beads as well as the glass slides were modified covalently with matching DNA-molecules. The patterned areas on the slides were defined by printing DNA-molecules with an optimized micro contact printing procedure using agarose gel stamps. In the following hybridization experiment the addressed beads self-assemble selectively on the matching areas of the glass substrate. Control experiments with mismatching DNA-oligonucleotides showed that silica beads tend to adhere strongly to the glass surfaces. Washing conditions must be controlled carefully to differentiate between hybridized beads and non-specifically bound beads. With regard to the use of this method in microelectronic chip assembly it could be shown that the salt concentration during the hybridization step can be reduced drastically without affecting the hybridization reaction.     

Artificial allosteric control of maltose binding protein

We demonstrate the allosteric control of a protein based on mechanical tension. When substrate binding is accompanied by a significant change of conformation of the protein, a mechanical tension favoring one or the other conformation will alter the binding affinity for the substrate. We have constructed a chimera where the two lobes of the maltose-binding protein are covalently coupled to the ends of a DNA oligomer. The mechanical tension on the protein is controlled externally by exploiting the difference in stiffness between single stranded and double stranded DNA. We report that the binding affinity of the protein for its substrates is significantly altered by the tension.     

Spectroscopic study on the interaction of eugenol with salmon sperm DNA in vitro

Fluorescence spectra, absorption spectra, melting temperature, ionic strength effect, and viscosity experiments were described that characterize the interaction of eugenol with salmon sperm DNA in vitro. Eugenol was found to bind but weakly to DNA, with binding constants of 4.23×10³, 3.62×10³ and 2.47×10³ L mol^?1 at 18, 28 and 38 °C respectively. The Stern–Volmer plots at different temperatures suggested that the quenching type of fluorescence of eugenol by DNA was a static quenching. Both the relative viscosity and the melting temperature of DNA were increased by the addition of eugenol. The changes of ionic strength had no affect on the binding. In addition, the binding constant of eugenol with single stranded DNA (ssDNA) was larger than that of eugenol with double stranded DNA (dsDNA). These results revealed that the binding mode of eugenol to DNA was intercalative binding. The thermodynamic parameters ΔH, ΔG and ΔS were also obtained according to the Van't Hoff equations, which suggested that hydrogen bond or van der Waals force might play an important role in a binding of eugenol to DNA. Based on the theory of the Förster energy transference, the binding distance between DNA and eugenol was determined as 4.40 nm, indicating that the static fluorescence quenching of eugenol by DNA was also a non-radiation energy transfer process.     

Bubbles in DNA by random force

We model single strand binding (SSB) proteins as agents exerting randomly oriented force on the bonds in DNA unzipping. The fluctuating force is found to unzip the double stranded DNA (dsDNA) via opening of bubbles along the chain.     

Measurement of the phase diagram of DNA unzipping in the temperature-force plane

We separate double stranded lambda phage DNA by applying a fixed force at a constant temperature ranging from 15 to 50 degrees C, and measure the minimum force required to separate the two strands. The measurements also offer information on the free energy of double stranded DNA (dsDNA) at temperatures where dsDNA does not thermally denature in the absence of force. While parts of the phase diagram can be explained using existing models and free energy parameters, others deviate significantly. Possible reasons for the deviations between theory and experiment are considered.     

Revealing interactions between polyaza pyridinophane compounds and DNA/RNA polynucleotides by SERS spectroscopy

Surface‐enhanced Raman scattering spectroscopy (SERS) in the near‐infrared region had been applied to study interactions of polyaza pyridinophanes with single stranded RNA and double stranded DNA and RNA polynucleotides. Studied compounds, PYPOD and PHENPOD, differed in the central aromatic moiety, pyridine and phenanthroline, respectively, which linked two cyclic amines. An intense scattering was obtained from molecules adsorbed onto the silver nanoparticles, showing nonlinear concentration dependence in the 6.5 × 10⁻⁸−6.5 × 10⁻⁵ M range. New bands in spectra of PYPOD/polynucleotide and PHENPOD/polynucleotide mixtures were assigned to vibrational modes of polynucleotide moieties involved in interactions. SERS spectra of both polyaza pyridinophanes with single stranded RNA polynucleotides indicated hydrogen bonding between the small molecules and the nucleic bases of poly A, poly C and poly U, whereas an interaction of only PYPOD with poly G was suggested. SERS spectra implied that both compounds bound into the minor groove of the helical poly dAdT–poly dAdT polynucleotide, while none of polyaza pyridinophanes interacted with the grooves of the poly dGdC–poly dGdC polynucleotide. Nevertheless, intensity ratios suggested intercalation of the phenanthroline moiety of the PHENPOD molecules into the double helix of the guanine‐cytosine polynucleotide. Spectral features also indicated binding of the PYPOD molecules within the major groove of the double stranded RNA analog. Copyright © 2014 John Wiley & Sons, Ltd.     

Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.     

Biophysical and electrochemical properties of Self-assembled noncovalent SWNT/DNA hybrid and electroactive nanostructure

DNA self-assembled hybrid nanostructures are widely used in recent research in nanobiotechnology. Combination of DNA with carbon based nanoparticles such as single-walled carbon nanotube (SWNT), multi-walled carbon nanotube (MWNT) and carbon quantum dot were applied in important biological applications. Many examples of biosensors, nanowires and nanoelectronic devices, nanomachine and drug delivery systems are fabricated by these hybrid nanostructures. In this study, a new hybrid nanostructure has been fabricated by noncovalent interactions between single or double stranded DNA and SWNT nanoparticles and biophysical properties of these structures were studied comparatively. Biophysical properties of hybrid nanostructures studied by circular dichroism, UV–vis and fluorescence spectroscopy techniques. Also, electrochemical properties studied by cyclic voltammetry, linear sweep voltammetry, square wave voltammetry, choronoamperometry and impedance spectroscopy (EIS). Results revealed that the biophysical and electrochemical properties of SWNT/DNA hybrid nanostructures were different compare to ss-DNA, ds-DNA and SWNT singly. Circular dichroism results showed that ss-DNA wrapped around the nanotubes through π-π stacking interactions. The results indicated that after adding SWNT to ss-DNA and ds-DNA intensity of CD and UV–vis spectrum peaks were decreased. Electrochemical experiments indicated that the modification of single-walled carbon nanotubes by ss-DNA improves the electron transfer rate of hybrid nanostructures. It was demonstrated SWNT/DNA hybrid nanostructures should be a good electroactive nanostructure that can be used for electrochemical detection or sensing.     

The molding of biological features using a flexible polymer mold

This paper examines two fabrication techniques (soft lithography and UV embossing) employed in the replication of various structures at the micron and single nanometer regimes. Stretched and assembled forms of double stranded (ds) DNA (16-3 μm in length) were adsorbed on coated silicon oxide. The collective results show that the resolution of all the thermally cured polymers improves significantly when the processing time between the stamp and the template is increased with s-PDMS demonstrating a lateral resolution of <10nm.     

Direct observation of large temperature fluctuations during DNA thermal denaturation

In this Letter, we report direct measurement of large low frequency temperature fluctuations in double stranded DNA when it undergoes a denaturation transition. The fluctuation, which occurs only in the temperature range where the denaturation occurs, is several orders more than the expected equilibrium fluctuation. It is absent in single stranded DNA of the same sequence. The fluctuation at a given temperature also depends on the wait time and vanishes in a scale of a few hours. It is suggested that the large fluctuation occurs due to coexisting denaturated and closed base pairs that are in dynamic equilibrium due to the transition through a potential barrier in the scale of 25-30kBT0 (T0=300 K).     

A fluorescence based assay for DNA damage induced by radiation,chemical mutagens and enzymes

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) radiation, chemical mutagens or restriction enzymes produced an assay response. UV radiation at 254 nm (approximating UV-C) and 360 nm (approximating UV-A) were used to induce the damage in dsDNA. Chemical damage was induced using several compounds with known effects on nucleic acids. Restriction enzymes Hind III, Msp1, Sau 3A1 were used to cut the plasmid (pUC19) at specific sequences in addition to the non-specific endonuclease DNase I. The effects of these types of damage on repeated melting and annealing of dsDNA were observed in real time using several fluorescence indicator dyes. Low concentrations of dsDNA (between 10 and 100 ng/ml) and small volumes (20 μl) were required for this assay. Repeated measures yielded a coefficient of variation of 2% (CV%). In addition to measuring various DNA damaging agents, the potential application of this assay to study the efficiency of various sun blocking agents against UV-induced DNA damage is discussed.     

Relaxation dynamics of semiflexible polymers

We study the relaxation dynamics of a semiflexible chain by introducing a time-dependent tension. The chain has one of its ends attached to a large bead, and the other end is fixed. We focus on the initial relaxation of the chain that is initially strongly stretched. Using a tension that is self-consistently determined, we obtain the evolution of the end-to-end distance with no free parameters. Our results are in good agreement with single molecule experiments on double stranded DNA.     

Magnetic Circular Dichroism Study of Analogues of the DNA Binding Ligands Netropsin and Distamycin A

UV absorption and MCD data of a complete series of netropsin-like oligopeptides containing between one and five N-methyl-pyrrole residues are reported. The presence of three MCD bands in the region between 220 and 350 nm can be related to the resonance of the peptide groups with the methylpyrrole systems of the oligopeptides. The longest wavelength absorption bands of all netropsin-like compounds investigated are ascribed to two electronic transitions of the resonance methypyrrole-carboxamide units. - Complex formation with DNA affects these MCD bands and hence modifies the transitions. This is explained by hypercon-jugation in the chromophoric system as a result of specific hydrogen bonding of peptide groups with dA·dT pairs of double stranded DNA.     

Characterization of the Binding of Adriamycin to Dna by Spectroscopic Methods

Adriamycin(ADM) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism, red shifts, and an isosbestic point in the absorption spectra were observed when ADM binds to calf thymus DNA(CT DNA), which suggested the intercalation mechanism of ADM into DNA bases. Upon binding to DNA, the fluorescence from ADM was efficiently quenched by the DNA bases, with no shifts in the emission maximum. the large increases in the polarization upon binding to CT DNA supported the intercalation of ADM into the helix. Iodide quenching studies showed that the magnitude of K_sv of the bound ADM was lower than that of the free ADM. the results of competitive binding studies showed that ethidium bromide could be displaced by Adm. Thermal denaturation experiments exhibited that the quenching of the fluorescence from ADM by single strand(ssDNA) was smaller than that by double strand(dsDNA). the results of all further studies also proved the intercalation of ADM into DNA base stack.