The formation of nanoconstructions based on double-stranded DNA molecules

The properties of nanoconstructions formed by double-stranded DNA molecules fixed in the structure of their liquid crystalline dispersions and cross-linked by nanobridges were described. The dependence of the optical properties of the nanostructures on various factors (DNA concentration, nanobridge components, etc.) was examined.     

Chromosomal stability and the DNA double-stranded break connection

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-stranded breaks in DNA are important threats to genome integrity because they can result in chromosomal aberrations that can affect, simultaneously, many genes, and lead to cell malfunctioning and cell death. These detrimental consequences are counteracted by two mechanistically distinct pathways of double-stranded break repair: homologous recombination and non-homologous end-joining. Recently, unexpected links between these double-stranded break-repair systems, and several human genome instability and cancer predisposition syndromes, have emerged. Now, interactions between both double-stranded break-repair pathways and other cellular processes, such as cell-cycle regulation and replication, are being unveiled.     

Self-assembly of double-stranded DNA molecules at nanomolar concentrations

Some proteins have the property of self-assembly, known to be an important mechanism in constructing supramolecular architectures for cellular functions. However, as yet, the ability of double-stranded (ds) DNA molecules to self-assemble has not been established. Here we report that dsDNA molecules also have a property of self-assembly in aqueous solutions containing physiological concentrations of Mg2+. We show that DNA molecules preferentially interact with molecules with an identical sequence and length even in a solution composed of heterogeneous DNA species. Curved DNA and DNA with an unusual conformation and property also exhibit this phenomenon, indicating that it is not specific to usual B-form DNA. Atomic force microscopy (AFM) directly reveals the assembled DNA molecules formed at concentrations of 10 nM but rarely at 1 nM. The self-assembly is concentration-dependent. We suggest that the attractive force causing DNA self-assembly may function in biological processes such as folding of repetitive DNA, recombination between homologous sequences, and synapsis in meiosis.     

The double-stranded DNA stability in presence of a flexible polymer

The mixture of the short segments of double-stranded DNA and a flexible polymer are addressed. It is shown that in the condensed phase, rigid DNA molecules exhibit transition between isotropic and orientationally ordered phases. It is shown that orientational ordering stabilizes the secondary structure of double-stranded DNA that could be relevant for the regulation of the gene expression at the condensed state of DNA.     

Selective trapping of circular double-stranded DNA molecules in solidifying agarose

When a solution containing agarose and DNA at 65 °C is allowed to solidify in the well of a preformed gel, it is found that circular DNAs become “trapped” in the newly formed matrix and resist electrophoretic migration. This finding provides an independent method for the characterization of circular DNA. The trapping phenomenon is dependent on the size, conformation, and concentration of circular DNAs as well as on the concentration of agarose. It is demonstrated that this technique can be used as a sensitive assay for detecting circular DNA.     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

Mechanical stability of single DNA molecules

Using a modified atomic force microscope (AFM), individual double-stranded (ds) DNA molecules attached to an AFM tip and a gold surface were overstretched, and the mechanical stability of the DNA double helix was investigated. In lambda-phage DNA the previously reported B-S transition at 65 piconewtons (pN) is followed by a second conformational transition, during which the DNA double helix melts into two single strands. Unlike the B-S transition, the melting transition exhibits a pronounced force-loading-rate dependence and a marked hysteresis, characteristic of a nonequilibrium conformational transition. The kinetics of force-induced melting of the double helix, its reannealing kinetics, as well as the influence of ionic strength, temperature, and DNA sequence on the mechanical stability of the double helix were investigated. As expected, the DNA double helix is considerably destabilized under low salt buffer conditions (相似文献   

Cleavage of double-stranded DNA by 'metalloporphyrin-linker-oligonucleotide' molecules: influence of the linker.

Manganese porphyrin-linker-triple-helix-forming oligonucleotide molecules were prepared and their ability to cleave in vitro a double-stranded DNA target present in the HIV-1 genome was studied. The nature of the linker is a determining factor of the cleavage efficiency. Cleavage yields as high as 80% were observed when the linker was a spermine residue and in the absence of a large excess of free spermine known to stabilize triplex structures. The hydrophobic nature of aliphatic diamine linker modified the cleaver-DNA interactions and reduced the efficiency of DNA cleavage.     

Liquid-crystalline dispersions formed by the complexes of linear double-stranded DNA molecules with dendrimers

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.     

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.     

The influence of low-energy millimeter electromagnetic waves on the stability of DNA molecules in solution

The influence of low-energy millimeter electromagnetic waves on aqueous saline solution of DNA from the liver of healthy rats and rats with sarcoma 45 has been investigated. The characteristic parameters of irradiated and unirradiated DNA, melting temperature, and the range of melting were obtained from melting curves. The duration of exposure did not practically affect the range of melting, while the thermostability of DNA increased; as irradiation duration increased to 90 min, the melting temperature of tumor increased by approximately 1.5 degrees C. It was assumped that the increase in the thermostability of DNA is due to a more effective stabilization of the DNA double helix caused by the dehydration of Na(+)- ions present in the solution.     

Temperature-induced changes of the packing of double-stranded linear DNA molecules in particles of liquid-crystalline dispersions

The circular dichroism spectra of liquid-crystalline dispersions obtained by phase exclusion of linear double-stranded DNA molecules from aqueous saline solutions of polyethylene glycol (120 ≤ C_PEG ≤ 300 mg/mL) have been investigated. The formation of liquid-crystalline dispersions at polyethylene glycol concentrations ranging from 120 to 200 mg/mL was accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism; this is indicative of cholesteric packing of the double stranded DNA molecules in the particles of the dispersion. Liquid-crystalline dispersions formed at PEG concentrations higher than 220 mg/mL and room temperature did not show any abnormal bands in the circular dichroism spectra; this is indicative of hexagonal packing of double-stranded DNA molecules in the particles of the dispersions. Heating of optically inactive liquid crystal dispersions induced a transition of the dispersions into a different state accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism. This transition is considered within the concept of the transformation of a hexagonal packing of DNA molecules into a cholesteric packing. A qualitative mechanism of such a transition is proposed that is formulated in the terms of the “quasinematic” layers of double-stranded DNA molecules that change their spatial orientation under the competing influences of the osmotic pressure of the solvent, orientational elasticity of the cholesteric packing, and thermal fluctuations.     

Hydration regulates the thermodynamic stability of DNA structures under molecular crowding conditions

Although water is an integral part of DNA structures, the effects of water molecules on various DNA structures which are formed by not only Watson-Crick but also Hoogsteen base pairs are still unclear. Here, we studied quantitatively the effects of molecular crowding on the thermodynamics of a parallel G-quadruplex formation of [d(TG 4)2]4 with Hoogsteen base pairs. It was demonstrated that molecular crowding conditions stabilized the parallel G-quadruplex. Moreover, the plot of stability of the parallel G-quadruplex structure versus water activity suggested that water molecules were released through the G-quadruplex formation. The stabilization of the DNA structures consisting of Hoogsteen base pairs under cell-like conditions may lead to a structural polymorphism of various DNA sequences regulated by water molecules.     

Length, mass, and denaturation of double-stranded RNA molecules compared with DNA

The contour lengths of linear, double-stranded (ds) RNAs from mycovirus PcV and Pseudomonas bacteriophage phi 6 have been measured with samples prepared for the electron microscope from 0.05 to 0.5 M NH4Cl solutions. A linear dependence of contour length on the logarithm of ionic strength was found and compared with that of dsDNA (pBR322, linearized and open-circular forms). Conditions for molecular weight determinations of any natural dsRNA by electron microscopy have been established, and the method has been calibrated with phi 6 dsRNA of known nucleotide sequence. The results imply that dsRNA in 0.20 M NH4Cl solution has a rise per basepair of 0.271 nm, which is shorter than that in the A-conformation (4%) and in the A'-conformation (10%). The thermal behavior of dsRNA in terms of melting temperature and exhibition of fine structure of melting curves was found to be generally similar to that of dsDNA, as expected from the literature. Folding of dsRNA in ethanolic solution was similar to that of dsDNA. However, in contrast to dsDNA, coiled coils could not be induced by ethanol, which is consistent with dsRNA being stiffer than dsDNA. Concerning dsDNA, the results show that a contraction in rise per basepair by 0.1 nm is coupled with an increase in the winding angle between basepairs by 0.47 degrees, as qualitatively predicted by polyelectrolyte theory.     

Structural polymorphism of the liquid-crystalline dispersions formed from the double-stranded DNA molecules complexed with synthetic polycations

The double-stranded, linear DNA molecules form the liquid-crystalline dispersions (LCD) in water-salt solutions containing positively charged polyconidin molecules. It was established from the analysis of the absorption spectra of the LCDs formed from (DNA-polyconidin) complexes, that the mean size of the particles of these dispersions is equal to -6000 angstroms. The small-angle X-ray data show, that in the LCD particles different density of packing of the (DNA-polycation) complexes is realized. The comparison of the X-ray data of the liquid-crystalline phases of (DNA-polyconidin) complexes formed under various conditions with the phase diagram, that reflects the polymorphism of the linear double-stranded DNA liquid crystals, demonstrates that the hexagonal mode of the LCD packing is existing in 0.15-0.4 M NaCl solutions, whereas in 0.4-0.55 M NaCl solutions-- the cholesteric one. As a result of specific spatial organization the cholesteric LCD possesses of an abnormal optical activity in the CD spectrum. The similar situation takes place in the case of another synthetic polycation--poly(2,5-ionen), whose chemical structure differs from that of polyconidin. Thus, the structural polymorphism of the (DNA-polyconidine) LCDs was evidenced. It means that change of NaCl concentration opens a gate to control the spatial packing of the molecules of (DNA-polycation) complexes in the particles of LCDs. The supposition about mechanism of formation of the DNA cholesteric liquid-crystalline state in the narrow interval of NaCl concentrations was suggested.     

Method enabling pyrosequencing on double-stranded DNA

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.     

Ligand effects on protein thermodynamic stability

A simple, partition-function formalism is used to describe the coupling between ligand binding and protein equilibrium unfolding. This general theoretical framework is shown to provide an adequate basis for the analysis of experimental ligand effects on the unfolding of complex protein systems. Nevertheless, the most important consequences of ligand binding for protein thermodynamic stability, as exposed by the partition-function approach, are found to be those demonstrated by Julian Sturtevant about 20 years ago.     

On the character of the thermodynamic properties distribution in DNA molecules inside the melting interval.

The variances of the distributions of DNA molecules over the degree of helicity and over the number of unwound regions inside the melting interval are calculated. The variance over the degree of helicity is expessed in terms of the values directly available from the experimental data. For the variance over the number of unwound regions a simple interpolation formula based on the machine calculations is proposed. Possible applications of the results obtained to interpolaation of the electron microscopic denaturation maps of DNA are discussed.