Changes Of DNA Molecules Research Articles

Telomere length, oxidative and epigenetic changes in blood DNA of patients with exacerbated psoriasis vulgaris

BackgroundThe pathogenesis of psoriasis vulgaris involves changes in DNA molecules, genomic instability, telomere attrition, and epigenetic alterations among them. These changes are also considered important mechanisms of aging in cells and tissues. ObjectiveThis study dealt with oxidation damage, telomere length and methylation status in DNA originating from peripheral blood of 41 psoriatic patients and 30 healthy controls. MethodsOxidative damage of serum DNA/RNA was determined immunochemically. Real-time PCR was used for the analysis of the telomere length. ELISA technique determined levels of 5-methylcytosine in blood cells' DNA. ResultsOxidative damage of serum DNA/RNA was higher in patients than in controls (median, 3758 vs. 2286pg/mL, p<0.001). A higher length of telomeres per chromosome was found in patients whole-cell DNA than in controls (3.57 vs. 3.04 kilobases, p=0.011). A negative correlation of the length of telomeres with an age of the control subjects was revealed (Spearman’s rho=-0.420, p=0.028). Insignificantly different levels of 5-methylcytosine in patients and controls were observed (33.20 vs. 23.35%, p=0.234). No influences of sex, smoking, BMI, PASI score, and metabolic syndrome on the methylation status were found. Study limitationsi) A relatively small number of the participants, particularly for reliable subgroup analyses, ii) the Caucasian origin of the participants possibly influencing the results of the parameters determined, and iii) Telomerase activity was not directly measured in serum or blood cells. ConclusionThe study demonstrated increased levels of oxidized DNA/RNA molecules in the serum of patients with exacerbated psoriasis vulgaris. The results were minimally influenced by sex, the presence of metabolic syndrome, or cigarette smoking. In the psoriatic blood cells' DNA, the authors observed longer telomeres compared to healthy controls, particularly in females. Insignificantly higher global DNA methylation in psoriasis cases compared to the controls indicated marginal clinical importance of this epigenetic test performed in the blood cells' DNA.

Biphasic Effect of Polyamines on Gene Expression in Relation to the Specific Change of the Higher-Order Structure of DNA

Polyamines are concerned with many important biological functions including the effect on the structure and function of genomic DNA. We have studied polyamine-induced higher-order structural changes of DNA molecules with a size larger than several tens of kilo base pairs. (1) We have also found that polyamines exert opposite effect, promotion and inhibition, for gene expression depending on their concentrations. (2) Here, we will report the results of our study by focusing the different effects among three structural isomers of tetravalent polyamines on the higher-order structure of DNA and also on gene expression. We have adapted the tetravalent polyamines; spermine (SP[343]), thermospermine (TSP[334]) and N4-aminopropylspermidine (BSP[3(3)4]), All of these polyamines are naturally present in living cells. Based on the measurements of single DNA observation with fluorescence microscopy, it became clear that these polyamines cause the transition of the higher-order structure from an elongated coil onto a compact state and that the potency for inducing DNA compaction is in the order TSP > SP > BSP. We have also measured the reaction kinetics of in vitro gene expression. It was found that all of these three polyamines exhibit promotion and inhibition at their low and high concentrations, respectively and that kinetic profile concerns neatly with the potentiality on the higher-order structural change. In this presentation, we will discuss these specific effects of the tetravalent polyamine isomers on the higher-order structure and activity of gene expression with the help of theoretical calculation in the interaction energy of these polyamines onto double-stranded DNA.

DNA-based surrogate indicator for sanitation verification and predict inactivation of Escherichia coli O157:H7 using vibrational spectroscopy (FTIR)

Washing and sanitation of fresh produce and food contact surfaces are vital steps in reducing the risk of foodborne illness. However, there is a lack of process control and verification tools to directly assess reduction of bacteria during a sanitation process. The overall goal of this study was to evaluate DNA as a biochemical surrogate indicator to assess sanitation process. Structural and chemical changes in DNA molecules immobilized on a membrane surface (DNA@Anodisc) and suspended in an aqueous solution (In-Liquid-DNA) were assessed using vibrational spectroscopy and chemometric analysis by a comparison between isolated DNA and the DNA in live Escherichia coli O157:H7 cells. The results of Fourier Transform Infrared (FTIR) illustrate DNA oxidation, fragmentation and conformational changes from double-stranded (ds) to single-stranded (ss) DNA. The PCA model was able to discriminate different groups of samples which were exposed to different concentrations of chlorine (non-lethal, sub-lethal, and lethal; 0, 2, 5, 10, and 15 ppm). PLSR model results showed that the degree of DNA oxidation could be quantified and used successfully to predict the chlorine concentrations and bacterial count. The regression coefficient for predicted vs measured chlorine concentrations and bacterial count were satisfying for all treatments (R2 > 0.96). The results also showed that the extent of oxidation and fragmentation of DNA was relatively higher for the In-Liquid-DNA, compared to the DNA@Anodisc, and E. coli. The results also suggest that the impact of the chlorine on the DNA@Anodisc and the DNA in the E. coli cells were similar compared to the In-liquid DNA. Overall, these studies illustrate the potential of DNA based biochemical surrogate indicator for sanitation process validation of food contact surfaces and fresh produce and demonstrate effectiveness of a chemometric spectral approach for these measurements.

Binding of platinum derivative, oxaliplatin to deoxyribonucleic acid: structural insight into antitumor action

Platinum-derived chemodrugs constitute an active class in cancer therapeutics. Besides being potent against various solid tumors, oxaliplatin has been recognized as the first platinum compound to be approved for the treatment of colorectal cancer. Structurally, oxaliplatin consists of a platinum metal complexed to oxalate and diaminocyclohexane (DACH) and exert its anticancer action by inhibiting DNA replication and transcription. The present study highlights the binding properties of oxaliplatin with calf thymus DNA using spectroscopic methods to comprehend its binding mechanism at molecular level to overcome associated cellular resistance and side effects. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic outcomes confirm that oxaliplatin is a covalent binding agent and also provide sequence specificity in DNA molecule. Infrared spectral results further indicate that oxaliplatin alkylates purine nitrogenous bases majorly guanine residues (G) in the major groove via formation of either interstrand or intrastrand guanine–guanine d(GpG) and guanine–adenine d(GpA) (N7 position) crosslinks accompanied with a slight external binding to sugar–phosphate backbone. Again, circular dichroism (CD) spectroscopic results suggest subtle conformational changes in DNA molecule due to its complexation with oxaliplatin and duplex attains an intermediate conformational state, having characteristics of both B- and C-forms. Further, a moderate binding strength of 4.12 ± 0.2 × 104 M−1 for the interaction has been estimated via ultraviolet–visible spectroscopy. The inferences obtained from these investigations are encouraging and can form the basis for further exploration in the field of rational drug development based on platinum compounds possessing preferential binding for nucleic acid with improved competence.Communicated by Ramaswamy H. Sarma

Laboratory Activity to Promote Student Understanding of UV Mutagenesis and DNA Repair.

Changes in DNA molecules are common, due to the effects of UV light and other external and internal mutagens. Cells have a variety of repair mechanisms which serve to maintain the accuracy of the genetic code. This activity includes a low-cost, safe and technically feasible experiment, which allows students to observe the effects of UV mutagenesis and DNA photorepair in the halophilc archaeon, Haloferax volcanii. An optional extension links this activity to topics of immediate concern to students – how exposure to UVC light contributes to skin cancer risk and the protective effects of sunscreen. Students design and carry out an experiment to test whether SPF 15 sunscreen increases the lethal exposure time for H. volcanii by a factor of 15. Throughout the activity, discussion questions engage students in actively thinking about the biological phenomena and experimental procedures and analysis. This activity is designed for students in college or university genetics, microbiology, or introductory biology courses as well as in high school honors biology courses. Teachers report that this activity was valuable in helping students understand mutagenesis and photorepair and in developing student skills in designing and analyzing experiments.

Single-molecule observation of prokaryotic DNA replication.

Replication of DNA requires the coordinated activity of a number of proteins within a multiprotein complex, the replisome. Recent advances in single-molecule techniques have enabled the observation of dynamic behavior of individual replisome components and of the replisome as a whole, aspects that previously often have been obscured by ensemble averaging in more classical solution-phase biochemical experiments. To improve robustness and reproducibility of single-molecule assays of replication and allow objective analysis and comparison of results obtained from such assays, common practices should be established. Here, we describe the technical details of two assays to study replisome activity. In one, the kinetics of replication are observed as length changes in DNA molecules mechanically stretched by a laminar flow applied to attached beads. In the other, fluorescence imaging is used to determine both the kinetics and stoichiometry of individual replisome components. These in vitro single-molecule methods allow for elucidation of the dynamic behavior of individual replication proteins of prokaryotic replication systems.

Asymmetric Illumination of Optically Anisotropic Beads for Detecting Rotational Motion

Many biological molecules, including RNA polymerases and ATP synthases, undergo rotational motion during their biological processes. Characterizing these processes at the single molecule level with high temporal and spatial resolution can reveal valuable information about their mechanical and dynamical properties. We are particularly interested in the dynamics of conformational changes of DNA molecules under torsion. Toward this long term goal we have developed a methodology that enables us to directly record the angular displacement of particles undergoing rotational motion by using asymmetric illumination of optically anisotropic beads. The method is implemented by illuminating a partially metal-coated bead with a laser beam coupled into the back side of the objective. The laser beam illuminating beads in the sample plane is oriented at an oblique angle to form asymmetric illumination. We observe that the scattering signal of the bead changes with the angular displacement of the coating on the bead relative to the illuminating laser beam. We use an optoelectronic system to detect scattering signal which is the direct high-speed measurement of the angular displacement of the bead. We conclude that our method is able to map angular displacements to electrical signal and we can determine the angular displacement of the biological molecule when conjugated to the bead. Our method obviates image acquisition and image processing procedures commonly used in previous studies, and it has the potential to significantly enhance the bandwidth of detection. We envision usage of this method in a range of biophysical measurements including magnetic trapping, tethered particle motion. We plan to use this rotational tracking method in combination with magnetic tweezers, a single-molecule technique that enables the application of torsional stress to twist single DNA molecules to extract high bandwidth torsional mechanical properties and dynamics of the molecules.

Applications of electrochemiluminescence resonance energy transfer between CdSe/ZnS quantum dots and cyanine dye (Cy5) molecules in evaluating interactions and conformational changes of DNA molecules

In this work, electrochemiluminescence resonance energy transfer (ECRET) between CdSe/ZnS quantum dot (QD) as the donor and cyanine dye (Cy5) as the acceptor in the conjugates consisting of QD, DNA and Cy5 was studied in detail. When a negative potential was applied to the conjugates immobilized on an Au electrode, QDs emitted a light with a maximum emission (λm) of 590nm or transfered energy to proximal ground-state Cy5 molecules in 0.1mol/L phosphate buffer (pH 7.4) containing 0.1mol/LK2S2O8 and 0.1mol/L KNO3. The excited state Cy5 molecules relaxed to their ground state by emitting a light with a λm of 675nm. ECRET between QD and Cy5 in the conjugates could be used to evaluate the interactions between DNAs and to measure the conformational changes of DNAs as well as the distances between groups in DNAs. For the ECRET system, ECRET efficiency was high due to high Cy5-to-QD ratio and low Förster distance.

Measuring interactions and conformational changes of DNA molecules using electrochemiluminescence resonance energy transfer in the conjugates consisting of luminol, DNA and quantum dot

A method based on electrochemiluminescence resonance energy transfer (ECRET) between luminol as the donor and CdSe/ZnS quantum dot (QD) for evaluation of the interactions between DNAs and measurement of the conformational changes of DNAs was developed. When a positive potential was applied to the conjugates consisting luminol, DNA and QD, ECRET between luminol molecules and QDs could be detected in 0.2mol/L Na2CO3–NaHCO3 buffer (pH 10) containing 1.0×10−2mol/L H2O2. In this case, luminol molecules emitted a light with a maximum emission (λm) of 460nm or transferred energy to proximal ground-state QDs. The excited state QDs relaxed to their ground state by emitting a light with a λm of 655nm. The ECRET between luminol and QD was used to evaluate interactions between DNAs and to measure conformational changes of DNAs.

Electroperturbation of DNA in Jurkat Cells Under Nanosecond Pulsed Electric Fields

Intense (5-30 MV/m) ultra-short (10-300 ns) pulsed electric fields (US-PEFs) have been demonstrated to interact with sub-cellular membranes and structures [Schoenbach et al., IEEE Trans. Diel. El. Ins., 14 (5): 1088-1109, 2007]. Investigating how cells respond to nanosecond pulses is essential for a) development of biomedical applications of US-PEFs, including cancer treatment, and b) better understanding of the mechanisms underlying such bioelectrical effects. T-lymphoblast Jurkat cells were exposed to a single pulse of 60 ns duration and 2.5 MV/m amplitude, without affecting cell growth and viability, to give insight into effects underlying interaction mechanisms. A cuvette-based exposure system, fed by a classical Blumlein pulse-forming network, was employed. A statistically significant reduction in DNA migration was detected immediately after US-PEF exposure by means of the alkaline comet assay [Zeni et al., Sensors, 8: 485-496, 2008]. This effect, associated with plasma membrane poration (YO-PRO-1 uptake) has been demonstrated to recover within 2 hours after pulse delivery (Figure 1), indicating that our exposure protocol targets the nucleus, affecting DNA structure. In our experimental conditions, a transient conformational change in DNA molecule can thus be hypothesized.

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

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

Microcontroller-driven fluid-injection system for atomic force microscopy

We present a programmable microcontroller-driven injection system for the exchange of imaging medium during atomic force microscopy. Using this low-noise system, high-resolution imaging can be performed during this process of injection without disturbance. This latter circumstance was exemplified by the online imaging of conformational changes in DNA molecules during the injection of anticancer drug into the fluid chamber.

In situ Study of DNA Attachment and Hybridization at Silicon Surfaces by Infrared Absorption Spectroscopy

In this study, we have investigated in situ the process of DNA immobilization and the subsequent hybridization on Si surfaces by using infrared (IR) absorption spectroscopy in the geometry of multiple internal reflection (MIR-IRAS). We use 3-aminopropyl-triethoxysilane (APTES) to functionalize Si surfaces with the amine group for DNA attachment. MIR-IRAS data, together with ab initio calculations, demonstrate that the amine-terminated surfaces are covalently coupled to thiol-modified oligonucleotides using a crosslinker of sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC). Hybridization experiments reveal that MIR-IRAS enables us to detect DNA hybridization through IR spectral changes in the frequency region around 1685 cm-1 where the vibrational modes of the bases appear. The present results show that MIR-IRAS is a promising tool for the label-free detection of DNA hybridization as well as the in situ (in vitro) characterization of the conformation changes of DNA molecules immobilized on Si surfaces.

DNA linearization through confinement in nanofluidic channels

Stretching DNA has emerged as a vital process for studying the physical and biological properties of these molecules. Over the past decade, there has been increasing research interest in utilizing nanoscale fluidic channels to confine and stretch single DNA molecules. Nanofabricated systems for linearizing DNA have revealed new and important insights into the conformation changes of DNA molecules. They also have emerged as innovative techniques for efficiently separating DNA molecules based on size and for physically mapping genetic information along the genome. This review describes physical theories of DNA linearization, current DNA stretching techniques based on nanofabricated channels, and breakthroughs resulting from the use of nanofluidic channels for DNA linearization.

Conformational changes of DNA molecules in interactions with bioactive compounds. II. DNA complexes with coordination compounds of cobalt and ruthenium

A comparative analysis is made of the character of conformational changes of the DNA molecule in its interaction with coordination compounds of cobalt and ruthenium with different ligand sets in the coordination sphere of the ion. It is shown that during complexation global conformational changes of DNA (e.g., the reduction of its volume) practically do not depend on the location and on the binding mode of the complex ion, but are primarily determined by its charge, whereas local conformational changes of the macromolecule are very sensitive to the type of the complex being formed, as indicated by the change of spectral properties of DNA. An assessment of sizes of the particles that are formed as a result of DNA condensation due to its binding with trivalent ions of cobalt and ruthenium was performed with the use of dynamic light scattering.

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.

Impact of Radiation Damage on DNA Determined by Computational Simulation

Review of molecular dynamics (MD) studies of several radiation-originated lesions on DNA molecules is presented. Main focus is to describe structural and energy changes in DNA molecule with the respect to proper recognition of the lesion by respective repair enzyme. Pyrimidine lesions (cytosinyl radical, thymine glycol) and purine lesion (8-oxoguanine) were subjected to MD simulations for several hundreds picosecond (ps), (200ps for cytosinyl radical, 2 nanosecond for thymine glycol and 8-oxoguanine) using MD simulation code AMBER 5.0 (4.0) and its respective force field modified for each lesion. Simulations were performed as all atoms simulations for fully solvated solute molecules in water. Negative charges of DNA phosphates were neutralized by sodium counterions Na+ that are essential for its double helical structure. In most cases the significant structural changes in DNA are observed: a) breaking of hydrogen bonds network between complementary bases and resulting opening of the double helix (cytosinyl radical, 8-oxoguanine); b) sharp bending of the DNA helix centered at the lesion site (thymine glycol); and c) flipped-out base (8-oxoguanine). These changes are related to overall collapsing of the double helical structure around the lesion and are considered to facilitate docking of the repair enzyme into the DNA and formation of DNA-enzyme complex. Stable DNA-enzyme complex is necessary condition for the onset of entire enzymatic repair process. In addition to the structural changes, specific values of electrostatic interaction energy are detected at several lesion sites (thymine glycol, 8-oxoguanine). The specific electrostatic energy is considered as a factor that enables repair enzyme to discriminate lesion from native, non-damaged site.

The effect of spermine on plasmid condensation and dye release observed by fluorescence correlation spectroscopy.

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHbetaAPr-1-neo (10 kbp; contour length 3.4 microm) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 microM (spermine/DNAphosphate=0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.0x10(-12) m2/s to 6.0x10(-12) m2/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dye's dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

Molecular dynamics simulation of thymine glycol-lesioned DNA reveals specific hydration at the lesion.

One nanosecond molecular dynamics (MD) simulation of a thymine glycol (TG)-lesioned part of human lymphoblast AG9387 was performed to determine structural changes in DNA molecule caused by the presence of a lesion. These changes can be significant for proper recognition of lesions by a repair enzyme. Thymine glycol is the DNA oxidative lesion formed by addition of OH radicals to C5 and C6 atoms of the thymine base. This lesion is known as causing Cockayne Syndrome-inherited genetic disorder. Distribution of water molecules in a hydration shell around the DNA molecule was analyzed for its contribution to the recognition of the TG lesion by the repair enzyme. The results of MD simulation show there is a specific DNA structural configuration formed at the lesion. After 500 ps the DNA is bent in a kink at the TG site. This change dislocates the glycosyl bond at C5' to a position closer to the DNA surface, and thus its atoms are more exposed to the surrounding water shell. The increased number of water molecules that are close to the TG site indicates that the glycosyl bond may be easily contacted by the repair enzyme. In addition, the higher number of water molecules at the TG site substantiates the importance of water-mediated hydrogen bonds created between the repair enzyme and the DNA upon formation of the complex. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1723-1731, 2001

Structural changes in DNA molecules induced by ultralow-intensity field of thermalized neutrons.

Structural changes in DNA molecules induced by ultralow-intensity field of thermalized neutrons.