DNA Aggregation Research Articles

核の力学的拘束・変形による核内DNAの分布・凝集の変化が細胞紫外線耐性に与える影響

核の力学的拘束・変形による核内DNAの分布・凝集の変化が細胞紫外線耐性に与える影響

微細加工基板を用いた細胞核内の DNA 分布・凝集操作に関する研究

微細加工基板を用いた細胞核内の DNA 分布・凝集操作に関する研究

Introduction of a tailor made anion receptor into the side chain of small peptides allows fine-tuning the thermodynamic signature of peptide-DNA binding.

The binding between peptides and DNA is often driven by entropic forces. We demonstrate herein a new approach to shift the thermodynamic profile of peptide/DNA binding from entropy to enthalpy driven. This eventually leads to higher compacted DNA aggregates which are important for gene transfection.

Morphology and properties of globular polymeric materials in the solid state: A composite material of DNA with a cationic surfactant

The aim of the present study was to control entanglements in order to regulate the properties of polymeric solids. Initially, fabrication of polymeric solids with few entanglements was attempted. Films of the DNA–cationic surfactant, cetyltrimethylammonium bromide (CTAB) (DNA–CTA), were cast from ethanol solution at room temperature. Morphological examination of DNA–CTA complex films using atomic force microscopy (AFM) revealed that these films were constructed by particle-like substances. Geometrical analysis of AFM images showed that the particle-like substances were the aggregates of several DNA–CTA globules. Mechanical characterization suggested that there were fewer entanglements than with normal plastic films. Small angle X-ray scattering experiments during annealing indicated that molecular motions were highly excited in the surface region of each particle. In conclusion, a globular polymeric film with fewer entanglements was fabricated. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2016, 54, 730–738

DNA Compaction and Particle Formation by Synthetic Polymers: Applications in Gene Delivery

We have reviewed the effects of synthetic polymers on DNA compaction and particle formation. Synthetic polymers such as poly (ethylene glycol) PEG-(PEG-3350, PEG-6000), methoxypoly (ethylene glycol) anthracene (mPEG-anthracene), methoxypoly (ethylene glycol) poly (amidoamine) (mPEG-PAMAM-G3), (mPEG-PAMAM-G4) and poly(amidoamine) (PAMAM-G4) alter DNA structure and dynamic. The spectroscopic results and atomic force microscopic (AFM) were analysed and the effect of synthetic polymer complexation on DNA stability, aggregation, compaction and particle formation are discussed. A comparison of the overall binding constants showed that the order of binding PAMAM-G4>PEG-6000>PEG-3350>mPEG-anthracene> mPEG-PAMAM-G4>mPEG-PAMAM-G3. The morphology and ultrastructure of polymer-DNA adducts showed major DNA compaction and particle formation induced by synthetic polymers. The generated information is useful for the application of synthetic nanoparticles in gene delivery.

Exonuclease-Catalyzed Target Recycling Amplification and Immobilization-free Electrochemical Aptasensor.

A simple, sensitive, and selective immobilization-free electrochemical aptasensor had been developed which combines the advantages of the discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide (ITO) electrode, high selectivity of the aptamer, and high efficiency of exonuclease-catalyzed target recycling amplification. Ochratoxin A (OTA), a type of mycotoxin, has been chosen as the model target. Methylene blue (MB) labeled probe DNA had been hybridized with the OTA aptamer first, which cannot diffuse freely to the negative charged ITO electrode surface due to the repulsion of the negative charges, since the hybridized DNA contains large negative charges. In the presence of target (OTA), the aptamer prefers to form an OTA-aptamer complex in lieu of an aptamer-DNA duplex, which results in the dissociation of probe DNA from the probe DNA-aptamer complex. The released probe DNA could be digested into mononucleotides, including a MB-labeled electroactive mononucleotide (eT), due to the employment of the RecJf exonuclease, a single-stranded DNA specific exonuclease. Since the eT contains little negative charge, it can diffuse easily to the negative charged ITO electrode surface, which results in the enhanced electrochemical response detected. At the same time, the aptamer in the OTA-aptamer complex can be digested by RecJf exonuclease also to liberate the target, which can participate in the next reaction cycling and realize the electrochemical signal amplification. Based on this strategy, an ultrasensitive homogeneous immobilization-free electrochemical aptasensor for OTA can be developed with a low detection limit (LOD) of 0.004 ng mL(-1) (S/N = 3). The proposed biosensor combines the advantages of the simplicity of immobilization-free homogeneous ITO based electrochemical determination, high efficiency of exonuclease-catalyzed target recycling, and high selectivity of the aptamer. The fabricated biosensor has been applied to detect OTA in real samples with satisfactory results. The same strategy can be applied to develop biosensors for diverse targets.

Structural modeling for DNA binding to antioxidants resveratrol, genistein and curcumin

Several models are presented here for the bindings of the antioxidant polyphenols resveratrol, genistein and curcumin with DNA in aqueous solution at physiological conditions. Multiple spectroscopic methods and molecular modeling were used to locate the binding sites of these polyphenols with DNA duplex. Structural models showed that intercalation is more stable for resveratrol and genistein than groove bindings, while curcumin interaction is via DNA grooves. Docking showed more stable complexes formed with resveratrol and genistein than curcumin with the free binding energies of −4.62 for resveratrol-DNA (intercalation), −4.28 for resveratrol-DNA (groove binding), −4.54 for genistein-DNA (intercalation), −4.38 for genistein-DNA (groove binding) and −3.84kcal/mol for curcumin-DNA (groove binding). The free binding energies show polyphenol-DNA complexation is spontaneous at room temperature. At high polyphenol concentration a major DNA aggregation occurred, while biopolymer remained in B-family structure.

Revealing Three Stages of DNA-Cisplatin Reaction by a Solid-State Nanopore.

The dynamic structural behavior in DNA due to interaction with cisplatin is essential for the functionality of platinum-based anti-cancer drugs. Here we report a novel method to monitor the interaction progress in DNA-cisplatin reaction in real time with a solid-state nanopore. The interaction processes are found to be well elucidated by the evolution of the capture rate of DNA-cisplatin complex, which is defined as the number of their translocation events through the nanopore in unit time. In the first stage, the capture rate decreases rapidly due to DNA discharging as the positive-charged hydrated cisplatin molecules initially bond to the negative-charged DNA and form mono-adducts. In the second stage, by forming di-adducts, the capture rate increases as DNA molecules are softened, appears as the reduced persistence length of the DNA-cisplatin adducts. In the third stage, the capture rate decreases again as a result of DNA aggregation. Our study demonstrates a new single-molecule tool in exploring dynamic behaviors during drug-DNA reactions and may have future application in fast drug screening.

Compaction and decompaction of DNA dominated by the competition between counterions and DNA associating with cationic aggregates

A systematic work concerning the DNA compaction and decompaction controlled by cationic surfactants, cetyltrimethylammonium with [FeCl3Br]− (CTAFe), Br− (CTABr) and Cl− (CTACl) as counterions, respectively, was performed. We discovered that cationic surfactants with complex counterions, [FeCl3Br]−, cannot promote the decompaction of DNA like those with Br− and Cl− as counterions. The rod-like CTAFe micelles were found to remain free in supernatants and cannot directly promote any redissolution or decompaction of DNA. These interesting findings could provide a better understanding of the interaction behavior of DNA and cationic surfactants. We conclude that the fundamental reason of the DNA decompaction lies upon the electrostatic competition between the counterions and DNA for associating with the cationic aggregates. At a high concentration, the binding of counterions to cationic CTA+ aggregates is promoted, which weakens and screens the electrostatic attraction between DNA and cationic aggregates. This could cause the decompaction of DNA as the cases of CTABr/DNA and CTACl/DNA mixtures. Our data revealed the fundamental reason of the compaction and decompaction behavior of DNA induced by cationic surfactants independently, a reasonable three-step model of the conformational changes of DNA controlled by different amounts of cationic surfactants was presented. The current work could provide a clear guidance in gene delivery, gene therapy and biomedicine fields.

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.

The peroxidase activity of ADM-Fe3+ cooperates with lipid peroxidation: The participation of hydroperoxide and hydroxyl radicals in the damage to proteins and DNA

To investigate the mechanisms of cardiotoxicity induced by adriamycin (ADM), the enzymatic activities of ADM-Fe3+, including the peroxidase and lipoxygenase (LOX) activity, and participation of active oxygen species in the damage to biological components were examined. ADM-Fe3+, but not ADM, steadily oxidized tetramethyl-p-phenylenediamine in the presence of peroxides, indicating that ADM-Fe3+ acts as a peroxidase. However, the activity of ADM-Fe3+ as peroxidase was very low compared with that of heme peroxidase, but was similar to that of LOX, which has a known peroxidase activity. Conversely, the activity of ADM-Fe3+ as a LOX was also very low compared with that of LOX itself. However, the lipid hydroperoxides (LOOH) produced by ADM-Fe3+ were the substrate for ADM-Fe3+ as a peroxidase. These findings indicate that lipid peroxidation cooperates with the peroxidase activity of ADM-Fe3+. Hydroxyl radicals (HO) were generated when ADM-Fe3+ was incubated with H2O2, but not with LOOH. Alcohol dehydrogenase was inactivated by LOOH. Conversely, DNA was mainly damaged by ADM-Fe3+ with H2O2. A small amount of DNA remained at the starting point on agarose gels during incubation with ADM-Fe3+ with LOOH and ADM-Fe3+ with H2O2. It seems that HO and compound I-like species participate in the strand breaks and the aggregation of DNA, respectively.

Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure.

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.

Single-molecule study on histone-like nucleoid-structuring protein (H-NS) paralogue in Pseudomonas aeruginosa: MvaU bears DNA organization mode similarities to MvaT.

Pseudomonas aeruginosa contains two distinct members of H-NS family of nucleoid-structuring proteins: MvaT and MvaU. Together, these proteins bind to the same regions of the chromosome and function coordinately in the regulation of hundreds of genes. Due to their structural similarity, they can associate to form heteromeric complexes. These findings left us wondering whether they bear similar DNA binding properties that underlie their gene-silencing functions. Using single-molecule stretching and imaging experiments, we found striking similarities in the DNA organization modes of MvaU compared to the previously studied MvaT. MvaU can form protective nucleoprotein filaments that are insensitive to environmental factors, consistent with its role as a repressor of gene expression. Similar to MvaT, MvaU filament can mediate DNA bridging while excessive MvaU can cause DNA aggregation. The almost identical DNA organization modes of MvaU and MvaT explain their functional redundancy, and raise an interesting question regarding the evolutionary benefits of having multiple H-NS paralogues in the Pseudomonas genus.

Detection of mercury ions (II) based on non-cross-linking aggregation of double-stranded DNA modified gold nanoparticles by resonance Rayleigh scattering method

This work describes a sensitive approach utilizing non-cross-linking aggregation of double-stranded DNA modified gold nanoparticles (dsDNA-AuNPs) for the detection of mercury ions (Hg2+) by resonance Rayleigh scattering (RRS) method for the first time. The double-stranded DNA contains a mismatched T–T base pair in the chain terminus, resulting in a flexible DNA tail and preventing the AuNPs from aggregation. Thus, a low RRS signal is obtained. However, in the presence of Hg2+, the non-cross-linking aggregation of dsDNA-AuNPs occurs, due to the Hg2+-mediated coordination of T–Hg2+–T base pair. The aggregation of nanoparticles generates a high RRS value. Particularly, the solution color and ultraviolet–visible absorption barely changed under the same conditions, while it is capable of detecting by RRS method with a low detection limit (0.4nM), which is 1000-fold lower than that of the colorimetric method. The proposed method was successfully applied to the detection of Hg2+ in real samples. The sensitive and selective assay might be inspiring for the development of new detectors for other metal ions or biomolecules.

Single molecular investigation of DNA looping and aggregation by restriction endonuclease BspMI.

DNA looping and aggregation induced by restriction endonuclease BspMI are studied by atomic force microscopy (AFM) and magnetic tweezers (MT). With Ca2+ substituted for the normal enzyme cofactor Mg2+ and enzyme concentration below the critical concentration of 6 units/mL, AFM images of DNA-BspMI complex show that the number of binding and looping events increases with enzyme concentration. At the critical concentration 6 of units/mL, all the BspMI binding sites are saturated. It is worth noting that nonspecific BspMI binding to DNA at saturation concentration represents more than 8% of the total BspMI-DNA complexes directly observed in AFM images. Furthermore, we used MT to prove that additional loops can form when enzyme concentration is higher than its saturation valueand the complex is incubated for a long time (>2 hrs). We ascribe this phenomenon to the aggregation of enzymes. The force spectroscopy of the BspMI-DNA complex shows that the pulling force required to open the loop of the complex at less than saturation concentration has a peak at about 3 pN, which is lower than the force required to open additional loops due to enzyme aggregation at higher than saturation concentration (>6 pN).

Lipid-modified polyethylenimine-mediated DNA attraction evaluated by molecular dynamics simulations.

The effect of lipid modification on polyethylenimine (PEI)-mediated DNA attraction was studied by performing umbrella sampling molecular dynamics simulations that involved PEIs modified with three different types of lipids: oleic acid (OA), linoleic acid (LA), and caprylic acid (CA). The potential of mean force between two DNA molecules in the presence of these lipid-modified PEIs was calculated using the weighted histogram analysis method, and it predicted the stability and size of the DNA aggregate. When compared to native PEI, lipid modification was found to enhance the stability of DNA aggregation in the case of long lipids (LA and OA) but reduce the stability in the case of a short lipid (CA). In addition, LA-substituted PEI was shown to form stronger DNA aggregate than OA-substituted PEI, which correlates positively with previous experimental observations.

Enhanced electrohydrodynamic collapse of DNA due to dilute polymers.

We experimentally demonstrate that addition of small, charge-neutral polymers to a buffer solution can promote compression of dilute solutions of single electrophoresing DNA. This phenomenon contrasts with the observed extension of DNA during capillary electrophoresis in dilute solutions of high molecular weight polymers. We propose these discrepancies in micron-scale DNA configurations arise from different nano-scale DNA-polymer collision events, controlled by solute polymer properties. We build upon theories previously proposed for intermolecular DNA aggregation in polymer-free solutions to develop scaling theories that describe trends seen in our data for intramolecular DNA compaction in dilute polymer solutions.

DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode

On the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide (ITO) microelectrode, a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed. Electroactive substance (methylene blue) tagged at the penultimate base T close to the 3'-terminal first, in the absence of DNA methylation, methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode. On the contrary, a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode. The proposed system does not need complex operation procedures such as bisulfite treatment, PCR amplification, and electrode immobilization. Six ITO microelectrodes had been assembled on the same microchip, which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening. The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity. The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Studies on Adsorption of DNA on Functional Ultrathin Films of Cationic Surfactant

We have investigated the interaction of DNA with a highly ordered functional ultrathin layer of cationic surfactant, dioctadecyl ammonium bromide (DOAB). The ultrathin film of DOAB is fabricated by Langmuir-Blodgett technique onto the pretreated quartz crystal wafers. The solution of DNA in phosphate buffer saline (PBS) is injected through a flow cell in a quartz crystal microbalance (QCM) loaded with the functional ultrathin film. The QCM data indicate a slower kinetics (time constant = 162.2 seconds) for the adsorption of DNA on DOAB layer as compared to PBS on DOAB. The surface morphology of the aggregation of DNA over the DOAB layer is investigated using atomic force microscope (AFM). The AFM image indicates the trapping of DNA over the DOAB layer. Such trapping of DNA can be potentially employed in the field of genomics.