Physicochemical Properties Of DNA Research Articles

DNA conformational flexibility study using phosphate backbone neutralization model.

Due to the critical role of DNA in the processes of the cell cycle, the structural and physicochemical properties of DNA have long been of concern. In the present work, the effect of interplay between the DNA duplex and metal ions in solution on the DNA structure and conformational flexibility is studied by comparing the structure and dynamic conformational behavior of a duplex in a normal form and its “null isomer” using molecular dynamics methods. It was found that the phosphate neutralization changes the cation atmosphere around the DNA duplex greatly, increases the major groove width, decreases the minor groove width, and reduces the global bending direction preference. We also noted that the probability of BI phosphate linkages increases significantly because of the charge reduction in the backbone phosphate groups. More importantly, we found that the electrostatic effect on the DNA conformational flexibility is dependent on the sequence; that is, the phosphate backbone neutralization induces the global dynamic bending to be less flexible for GC-rich sequences but more flexible for AT-rich sequences.

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.

Biophysical study of novel oligoelectrolyte‐based nonviral gene delivery systems for mammalian cells

Gene therapy is an important treatment for genetic and acquired diseases. The success of gene therapy is largely dependent on the development of suitable vectors for gene transfer. Vectors are needed to overcome cellular barriers and to achieve efficient DNA delivery with low cytotoxicity. In the present study, we synthesized and characterized a novel comb-like oligoelectrolyte nanocarrier, BG-2, as a nonviral gene delivery vector. A novel surface-active oligoelectrolyte of comb-like structure was synthesized via controlled radical copolymerization using oligoperoxide Cu(+2) coordinating complex as a multisite initiator of graft copolymerization. The critical micellar concentration was determined by Nile Red fluorescence. Complex formation of DNA with BG-2 was determined by YOYO-1 fluorescence. The physicochemical properties of DNA in complex with BG-2 have been investigated by electrophoresis, dynamic light scattering and fluorescence spectroscopy. The BG-2/DNA complex was demonstrated by scanning electron microscopy. Interactions between BG-2/DNA complex and model membranes were also studied. The sensitivity of the DNA molecule, complexed with BG-2, against deoxyribonuclease I and serum nucleases was assessed by agarose gel electrophoresis. BG-2 efficiency in the transfection of HeLa cells was determined by measuring luciferase gene expression using a luminometer and cytotoxicity was also evaluated. BG-2 oligoelectrolyte was successful in overcoming cellular barriers as a result of forming stable and small sized complexes with DNA, interacting with model membranes in a desirable manner and protecting DNA from nuclease. The transfection efficiency was quite high and cytotoxicity was low. BG-2 appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties.

Binding of Nucleoid-Associated Protein Fis to DNA Is Regulated by DNA Breathing Dynamics

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.

Alkylhydroxybenzenes modify immune complex size

Alkylhydroxybenzenes (AHBs) are a large group ofbiologically active substances of microbial and plantorigin that are capable of nonspecific interaction withdifferent biopolymers [1–7]. AHB homologues differin the length of the alkyl radical, which determines thedegree of hydrophobicity of the molecules and theirreactivity [1, 2]. The functioning of AHBs as “chemical chaperones” [3, 4] determines the development ofeffects in different systems. For example, changes inthe presence of micromolar concentrations of AHBswere shown for the catalytic activity of some enzymes[3, 4], the physicochemical properties of DNA [5],and the antigenbinding ability and functional activityof antibodies [6, 7]. It was also shown that the molecular weight and sizes of proteins modified with AHBsaltered [3]. However, information about the effect ofAHB on the dimensional characteristics of immunecomplexes is missing.MATERIALS AND METHODSSuspensions of model immune complexes wereformed from purified tetanus toxoid (400 EU/mL) andhuman antitetanus γglobulin containing not less than95% IgG of the total concentration (10%) of protein inthe sample. The study was performed at antigen/antibody ratios of 1 : 32/1 : 8, 1 : 16/1 : 2, 1 : 32/1 : 2, and1 : 64/1 : 2. We used chemical analogues of microbialAHBs (99.9% purity) differing by the length of thealkyl radical: C

Inferring Phylogenies from Physico-Chemical Properties of DNA

Phylogenetic analysis relies on alignment of related sequences from different species to obtain the distances between these species. The quality of the alignment and the distance measure would depend on the alignment parameters that are used. In this work, we propose to use Relative Complexity Measure (RCM) method to find the distances between the sequences which is a parameter independent measure. We used DNA sequences from Candida species and phylogenetic trees were obtained using un-weighted pair-group with arithmetic mean method. We used three reduced alphabets for the DNA sequences which were clustered by taking into account different physico-chemical properties of DNA. RCM gives as good results as the distance determination method and among the physico-chemical properties, Keto/Amino grouping is found to give the most accurate tree which is topologically closest to the desired phylogeny.

High DNA melting temperature predicts transcription start site location in human and mouse

The accurate computational prediction of transcription start sites (TSS) in vertebrate genomes is a difficult problem. The physicochemical properties of DNA can be computed in various ways and a many combinations of DNA features have been tested in the past for use as predictors of transcription. We looked in detail at melting temperature, which measures the temperature, at which two strands of DNA separate, considering the cooperative nature of this process. We find that peaks in melting temperature correspond closely to experimentally determined transcription start sites in human and mouse chromosomes. Using melting temperature alone, and with simple thresholding, we can predict TSS with accuracy that is competitive with the most accurate state-of-the-art TSS prediction methods. Accuracy is measured using both experimentally and manually determined TSS. The method works especially well with CpG island containing promoters, but also works when CpG islands are absent. This result is clear evidence of the important role of the physical properties of DNA in the process of transcription. It also points to the importance for TSS prediction methods to include melting temperature as prior information.

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a β-galactosidase reporter plasmid DNA applied to ViaDerm™ treated skin. Skin treated with ViaDerm™ using 50 μm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm™ was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm™) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.

Mechanisms of Interaction between DNA and Chemical Analogues of Microbial Anabiosis Autoinducers

The alkylhydroxybenzene (AHB) autoregulatory factors d1 (fd1) of microorganisms have been found to directly interact with highly polymeric DNA. This circumstance results in changes, related to alterations in the topology of this macromolecule, in DNA physicochemical properties. The physicochemical properties of DNA in the presence of chemical analogues of microbial AHBs (methylresorcinol; hexylresorcinol; and 2-(4-hydroxyphenyl)ethane-1-ol, also known as tyrosol) were investigated using adsorption spectrophotometry, fluorometry, heat denaturation, viscosimetry, and electrophoresis in agarose gel. A number of concordant effects pointing to DNA-AHB interactions were revealed that manifesed themselves in the hypochromic properties of the resulting complexes, an increase in their melting temperature and viscosity, a decrease in their electrophoretic mobility, and a change in the fluorescent properties of AHBs upon complexation with DNA. Such alterations were particularly significant in the presence of hexylresorcinol, which possessed the maximum alkyl radical length among the fd1 analogues tested. Using atomic force microscopy, we visualized the micelle-like DNA nanostructures forming in the presence of AHBs. The results obtained provided the basis for developing a hypothetical model of the interaction between the biopolymer macromolecule and low-molecular-weight AHBs that takes into account the differences in the hydrophobicity of individual AHB homologues functioning as ligands. In terms of our model, we discuss AHB involvement in the stabilization of DNA and alteration of its topology, i.e., in the process related to intragenomic rearrangements, which account for the intrapopulational variability of bacteria, including dissociation processes.

Preparation and phase behaviour of surface-active pharmaceuticals: self-assembly of DNA and surfactants with membranes. Differential adiabatic scanning microcalorimetric study

Some energetics issues relevant to preparation and surface characterization of zwitterionic phospholipid–DNA self-assemblies, as alternative models of the currently used problematic lipoplexes are presented. Nucleic acid compaction capacities of Mg 2+ and N-alkyl- N, N, N-trimetylammonium ions (C n TMA, n = 12) were compared, with regard to surface interaction with unilamellar vesicles. Differential adiabatic scanning microcalorimetric measurements of synthetic phosphatidylcholine liposomes and calf thymus DNA and their ternary complexes with Mg 2+ and C 12TMA, were employed for deduction of the thermodynamic model describing their structural transitions. Small monodisperce and highly stable complexes are established after precompaction of DNA with detergent, followed by addition of liposomes. In contrast, divalent metal cation-mediated aggregation of vesicles either leads to heterogeneous multilamellar DNA–lipid arrangements, or to DNA-induced bilayer destabilization and lipid fusion. Possible dependence of the cellular internalization and gene transfection efficiency on the structure and physicochemical properties of DNA–Mg 2+–liposomes or DNA–cationic surfactant–liposome systems is emphasized by proposing the structure of their molecular self-organizations with further implications in gene transfer research.

Study of ethidium bromide effect on dsDNA separation by capillary zone electrophoresis and laser light scattering.

The effect of ethidium bromide (EB) on dsDNA separation was studied by using capillary zone electrophoresis (CZE) and laser light scattering (LLS). By increasing the concentration from 1 to 15 microg/mL, EB showed significant influence on the separation of pBR322 HaeIII in terms of resolution, speed, and signal-over-noise ratio. The physicochemical properties of DNA, such as mobility under electric field, charge-over-size ratio, and hydrodynamic radius, showed two declines, a sharp drop followed by a relaxed decrease, with increasing EB concentration. Such observations could be explained by using two modes of the dye binding process: intercalation and outside binding. The combination of results obtained from CZE and LLS also confirmed the existence of outside binding. Compared with intercalation, outside binding showed a weaker dye concentration dependence in neutralizing the negative charges on the DNA fragments. Static light scattering showed that <M(w)> and <R(g)> of DNA in 1xTris-borate-EDTA (TBE) were increased with increasing EB concentration. <R(g)>/<R(h)> of DNA with dye had a value larger than 1.5, suggesting a random coil conformation.

DNA conformation and configuration in protein-DNA complexes: Current Opinion in Structural Biology 1992, 2:71…-77

The local conformations adopted by DNA in DNA-protein complexes are to a large extent consistent with the sequence-dependent variability in the physicochemical properties of DNA. The alteration of the configuration of DNA by certain DNA-bending proteins indicates that DNA curvature may play an important biological role in the enhancement of the rate of transcription initiation.

Comments on Commentary by D. Billen

Billen (1) proposes that the spontaneous to DNA due primarily to the thermodynamic instability arising from storage in the living genome at 37?C can be used to determine a negligible for ionizing radiations. The concept of DNA instability is, I believe, of great relevance to radiobiology, but logically it leads to a rather different conclusion, namely that however small the dose and however low the dose rate there is a finite chance that a biological effect will be caused. The concept of negligible does not belong in the domain of the physicochemical properties of DNA but in the domain of the acceptability of risk, a sociopolitical issue. My argument is as follows. I agree with Billen (1) that about 100 events occur in the genome of the diploid mammalian cell per minute and that cells have evolved efficient repair processes to enable them to cope with this rate of induction. The nature of these sites in double-stranded DNA, the repository of the genetic information, is base loss, base damage, and single-strand breaks, i.e., single-strand damage. While this is being continually produced it must be equally continually being repaired; i.e., there is a dynamic equilibrium between degradation and repair in the living cell. Even at this rate of formation the distances between the transitory lesions are enormous, and so the generation of double-strand due to the coincidence of single-strand sites in opposing strands is indistinguishable from zero. Cells do generate double-strand breaks; however, this is not primarily but rather part of their normal functioning and a controlled process. Radiation induces both singleand double-strand and DNA-protein crosslinks (2). I think, as does Billen, that provided the single-strand is not induced at a rate comparable to the rate of induction of spontaneous single-strand then the continually active repair processes will satisfactorily repair both the radiation-induced and the spontaneous damage. This is then irrelevant to biological effect. However, the double-strand (mainly breaks) and the DNA-protein crosslinks caused by radiation will not necessarily be repaired effectively, and it is these lesions that can constitute relevant damage and lead to biological effects at low dose rates. Thus I would predict that at low dose rates, radiation-induced biological effects would be directly proportional to dose and invariant with dose rate. At dose rates where the ate of induced single-strand was comparable with that ue to spontaneous I would predict a transition to a dependence of effect on dose rate due to the disruption of the dynamic equilibrium between DNA degradation and repair and the consequent build-up of unrepaired of both spontaneous and radiolytic origin. Indeed, such a transition is exactly what is observed for the induction of specific locus mutations in mice when males are irradiated at the spermatogonial stage (to be published elsewhere). At dose rates of 0.015 Gy/min (a singlestrand induction rate of about one-third that due to spontaneous damage) a transition is observed; below this rate mutation frequency is invariant with dose rate, and above this rate it increases sharply with increasing dose rate. Viewed in this way I believe the concept of DNA instability is of considerable value to radiobiology and radiological protection in the following ways:

Action at a distance along a DNA.

A number of ways are known by which an event at one location on a DNA molecule can affect an event at a distant location on the same molecule. Three classes of mechanisms are described for such distal actions: tracking or translocation of a protein along a DNA, the association of two proteins bound at separate sites to form a DNA loop in between, and distal interactions that are affected by the topology of the DNA. The basic characteristics of each type of mechanism are discussed in terms of the known physicochemical properties of DNA. The various modes of action at a distance are often interrelated. Examples include the formation of positively and negatively supercoiled DNA loops by tracking and the strong effects of DNA topology on looping.

Effect of enzymatic methylation on the chemical, physical, and biological properties of DNA

DNA methylase was partially purified from Escherichia coli W and used to methylate DNA from Bacillus subtilis and bacteriophage φ105. The former DNA was methylated 1.17% and the latter 0.87%. The products were 6-methyladenine (85%) and 5-methylcytosine (15%) in both cases. The methylated DNA was stable toward depurination and viscosity loss at elevated temperatures. Methylation led to a 50% decrease in transforming activity in two strains of B. subtilis and no change in a third strain. The ability of phage φ105 DNA to rescue a defective phage strain was decreased 50% by methylation. No changes were observed in the ability of methylated DNA to serve as a template for DNA polymerase or RNA polymerase. The pattern of cleavage of DNA by a variety of restriction endonucleases was not affected by methylation. There were no changes in the physicochemical properties of DNA on methylation as measured by hyperchromicity on heating, formaldehyde denaturation, viscosity, and sedimentation.

Calcium metabolism in tumors--its relationship with chromium complex accumulation. V. The interaction nucleic acid-calcium and the calcification of tumors.

The interaction nucleic acid-calcium was studied <i>in vitro</i>. RNA is precipitated by CaCl₂ while DNA is not. The presence of complexed chromium affects the precipitation of RNA and the physicochemical properties of DNA. The study of the cell fractions from experimental tumors indicate variations in the fraction distribution of calcium and nucleic acids related to the growth stage. The possible role of the cell membranes in the calcification process appears emphasized by the accumulation of calcium and nucleic acids in the microsomal fraction.