Surface-directed DNA condensation in the absence of soluble multivalent cations.

Multivalent cations are known to condense DNA into higher ordered structures, including toroids and rods. Here we report that solid supports treated with monovalent or multivalent cationic silanes, followed by removal of soluble molecules, can condense DNA. The mechanism of this surface-directed condensation depends on surface-mobile silanes, which are apparently recruited to the condensation site. The yield and species of DNA aggregates can be controlled by selecting the type of functional groups on surfaces, DNA and salt concentrations. For plasmid DNA, the toroidal form can represent >70% of adsorbed structures.     

General properties of silated hydroxyethylcellulose for potential biomedical applications.

The general properties of hydroxyethylcellulose (HEC) grafted with 3-glycidoxypropyltrimethoxysilane (GPTMS) or 3-glycidoxypropylmethyldiethoxysilane (GPDMS) were studied for potential biomedical applications. The graft involved a Williamson reaction between the free hydroxyl function of HEC and the epoxy function of the two silanes. As the grafted silanes are in ionic form (sodium silanolate), this product remains in gel form at basic pH (>12.3) in aqueous solution. When pH decreases, sodium silanolate is transformed into silanol (2 or 3 silanol functions are carried by silicon, depending on the silane grafted). The silanols interreact, and the gel is transformed into a cross-linking form at room or body temperature. Studies were conducted to optimise this product for specific uses. Steam sterilization was used to compare self-hardening as a function of the silane grafted. Our previous work indicated that HEC grafted with GPTMS has good reactivity, but requires high pH for dissolution, whereas dissolution occurs at lower pH with GPMDS. The rate of silanol condensation for silated HEC was then determined as a function of pH, temperature, type of silane, and the percentage grafted. Condensation rates were ascertained by the viscosity method, and gels were neutralized by different solutions to obtain buffered forms at various pH. The time required to obtain 10(5) mPa x s, with an initial state of 2500 mPa x s, was then calculated. Condensation was catalysed in acid or basic medium at a lower rate at pH 5.5-6.5, and a temperature rise increased the condensation rate, regardless of the pH or silane studied. Silanetriol was more reactive than silanediol. However, as HEC lost considerable viscosity after sterilization, further studies will be conducted to develop new polysaccharides grafted with silane.     

Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active.

The interconversion between the right (R) and left (L) helical forms of poly[d(G-C)] occurs at low concentrations of MgCl2 and EtOH, acting together in a highly synergistic manner. Thus, the cooperative R---L transition is induced by only 0.4 mM and 4 MM MgCl2 in combination with 20% and 10% EtOH, respectively. The L form of poly[d(G-C)] formed under these conditions has the spectroscopic properties (absorption, circular dichroism) previously demonstrated under high salt conditions (Pohl and Jovin, 1972) and thought to correspond to the left-handed Z DNA structures recently established by X-ray crystallography (Wang et al., 1979; Drew et al., 1980). However, L DNA formed in Mg2+-EtOH (which we designate as Z* DNA) has unique properties: a) it can be sedimented readily out of solution at low speed, indicative of condensation and intermolecular aggregation; b) it supports the binding of several intercalating (ethidium bromide, actinomycin D) and non-intercalating (mithramycin) drugs, although these interact preferentially with the R (i.e., B) form of DNA; and c) it functions as a template for Escherichia coli RNA polymerase. B and Z* DNAs can be generated under identical ionic conditions and compared in a number of biochemical systems. Our results suggest that left-handed DNA may form under physiological conditions and serve a biological function.     

The condensation of DNA by chromium (III) ions

Cr(III), one of the most potent inorganic carcinogens, induces condensation of DNA into a very compact product at 37 degrees, as shown by electron microscopy. The condensation begins with the appearing of some supercoil structures and complete condensation occurs at relatively low Cr(III) concentrations; for 3 and 30 mM ionic strength they are 4.5 and 45 microM, respectively. Under these conditions, Cr(III) inhibits the interaction between ethidium and DNA as shown by absorption and fluorescence spectra.     

Atomic force microscopy reveals the assembly of potential DNA "nanocarriers" by poly-L-ornithine

Atomic force microscopy (AFM) has been used to visualize the process of condensation of plasmid DNA by poly-L-ornithine on mica surface. AFM images reveal that the transition of negatively charged DNA to condensed nanoparticles on addition of increasing amounts of positively charged poly-L-ornithine (charge ratio (Z+/Z-) varied between 0.1 and 1) at a wide range of DNA concentrations (3-20 ng/microl) occurs through formation of several distinct morphologies. The nature of the complexes is strongly dependent on both the charge ratio and the DNA concentration. Initiation of condensation when the concentration of DNA is low (approximately 3-7 ng/microl) occurs possibly through formation of monomolecular complexes which are thick rod-like in shape. On the contrary, when condensation is carried out at DNA concentrations of 13-20 ng/microl, multimolecular structures are also formed even at low charge ratios. This difference in pathway seems to result in differences in the extent of condensation as well as size and aggregation of the nanoparticles formed at the high charge ratios. To the best of our knowledge, this is the first direct single molecule elucidation of the mechanism of DNA condensation by poly-L-ornithine. Cationic poly-aminoacids like poly-L-ornithine are known to be efficient in delivery of plasmid DNA containing therapeutic genes in a variety of mammalian cell lines by forming condensed "nanocarriers" with DNA. Single molecule insight into the mechanism by which such nanocarriers are packaged during the condensation process could be helpful in predicting efficacy of intracellular delivery and release of DNA from them and also provide important inputs for design of new gene delivery vectors.     

The Condensation of DNA by Chromium (III) Ions

Abstract

Cr(III), one of the most potent inorganic carcinogens, induces condensation of DNA into a very compact product at 37°, as shown by electron microscopy. The condensation begins with the appearing of some supercoil structures and complete condensation occurs at relatively low Cr(III) concentrations; for 3 and 30 mM ionic strength they are 4.5 and 45 μM, respectively. Under these conditions, Cr(III) inhibits the interaction between ethidium and DNA as shown by absorption and fluorescence spectra.     

Fluorescence microscopy of the dynamics of supercoiling, folding, and condensation of bacterial chromosomes, induced by acridine orange

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and supercoiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA, and indicate that kinks are an important intermediate step in many of the transitions.     

The observation of the local ordering characteristics of spermidine-condensed DNA: atomic force microscopy and polarizing microscopy studies.

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.     

Fluorescence Microscopy of the Dynamics of Supercoiling,Folding, and Condensation of Bacterial Chromosomes,Induced by Acridine Orange

Abstract

The fluorescent dye, acridine orange, was used to visualize bacterial chromosomes extending from bacteria attached to a glass surface. The acridine-induced condensation of these chromosomes was followed in real-time with a low light level video camera. Acridine orange induced the packing of the bacterial chromosome into thick bundles which underwent various forms of condensation, supercoiling, folding, and rolling into a compact particle. Filaments attached to the surface at both ends were topologically constrained and super- coiled rapidly; whereas all three patterns of condensation were noted among filaments attached at only one end or free from the surface. Kinks often appeared in the filaments prior to supercoiling or folding, and the dynamic events observed often occurred around these kinks. These observations identify several mechanisms of condensation available to higher order structures of DNA and indicate that kinks are an important intermediate step in many of the transitions.     

Structure and stability of sodium and potassium complexes of dT4G4 and dT4G4T.

The ends of eukaryotic chromosomes contain specialized structures that include DNA with multiple tandem repeats of simple sequences containing clusters of G on one strand, together with proteins which synthesize and bind to these sequences. The unit repeat in the protozoan Oxytricha with the cluster dT4G4 can form structures containing tetrads of guanine residues, referred to G4 DNA, in the presence of metal ions such as Na+ or K+. We show here that, in the presence of Na+, dT4G4 forms a tetramer with parallel strands by means of a UV cross-linking assay. In the presence of K+, two further interactions are observed: at low temperature, higher order complexes are formed, provided the 3' end of the strand is G; a single 3'T inhibits this association in dT4G4T. At high temperature, these complexes dissociate, leading to a tetramer with a different ordered structure that melts only at very high temperatures. These results suggest that the cohesive properties of DNA containing G clusters might depend on associative interactions driven by a free 3'G terminus in the presence of K+, as well as by connecting antiparallel G hairpins as has been postulated.     

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.     

DNA-binding properties of the major core protein of adenovirus 2.

The major adenovirus core protein (P.VII) binds to various species of duplex and single-stranded DNA molecules as a linear function of P.VII concentration. P.VII progressively condenses 32S Ad2 DNA into rapidly sedimenting forms having an S value of around 2,280. P.VII does not coat DNA like cytochrome C, instead DNA-protein beads are visualized in the electron microscope at low protein concentration. These beads appear to interact forming larger structures and at high P.VII concentrations the DNA molecule becomes highly compacted. Analysis of DNA fragments formed after digestion of P.VII-DNA complexes and isolated cores with micrococcal nuclease suggest that the organization of the DNA in the two structures is essentially identical. The initial P.VII and DNA interaction is sensitive to both ionic and hydrophobic environments, whereas the in vitro DNA-P.VII complexes are extremely stable and are not disrupted in the presence of 3 M NaCl, 1% sarcosyl or 5% deoxycholate. Properties of these in vitro DNA-protein VII complexes share striking similarities to isolated viral core particles.     

Pseudomonas bacteriophage phi KZ contains an inner body in its capsid

Electron microscopical examination of the new virulent bacteriophage phi KZ, specific for Pseudomonas aeruginosa, has revealed an unusual structure in its capsid. In the center of the phage head is a cylinder of low electron density ("inner body"), surrounded by fibrous material which is packed around the inner body in a spoollike manner. The inner body itself has a springlike appearance. These structures disappear after adsorption of phage particles to bacteria. Various morphological forms, which can be interpreted as intermediate steps in phi KZ DNA condensation, have been seen in ultrathin sections of phi KZ-infected cells.     

A direct demonstration that the ethanol-induced transition of DNA is between the A and B forms: an X-ray diffraction study

DNA undergoes a reversible co-operative change in a number of its properties over a characteristic range of ethanol concentrations. This ethanol-induced transition of DNA has been studied by a number of groups, using circular dichroism as well as other assay techniques. There has been disagreement as to the species of DNA involved. We have used X-ray diffraction methods on DNA fibers exposed to an excess of solvent. The diffraction patterns serve as “fingerprints” to clearly show that the transition occurs between the B and A forms of DNA (at low and high ethanol concentrations, respectively) in agreement with the conclusions of some of the previous workers. At still higher ethanol concentrations, the diffraction pattern changes from that of the A form to that of a more disordered form.     

Linking number anomalies in DNA under conditions close to condensation

Changes in linking number and the apparent winding angle of pBR322 DNA have been evaluated in mixed ethanol-water solvents containing either Na or Mg as the major counterion contributing to the electrostatic shielding of the duplex. The average number of superhelical turns (tau) produced in the standard electrophoresis buffer (Tris-borate-EDTA, pH 8.0) by the transfer of DNA, relaxed in 200 mM NaCl, 10 mM NaH2PO4/Na2HPO4, and 2 mM EDTA, pH 7, by calf thymus topoisomerase or ligated in 6.6 mM MgCl2, 1 mM KCl, 1 mM ATP, 1 mM dithiothreitol, and 66 mM Tris, pH 7.6, by T4 ligase, was determined as a function of the EtOH concentration. At low enzyme concentrations, the tau values became increasingly more positive in the presence of both cations as the ethanol concentration increased, indicating that the duplex structure was overwound in the ethanol solvents. Winding angle changes between 0 and 20% ethanol, calculated from these values of tau, exhibited the same correlations with CD spectral properties as had been previously observed for 100% aqueous systems containing monovalent cations [Kilkuskie, R., Wood, N., Shinn, R., Ringquist, S., & Hanlon, S. (1988) Biochemistry 27, 4377-4386]. The results at higher concentrations of ethanol (25-30%), however, were anomalous for the Mg-ligase system. The anomalies increased with higher ethanol, ligase, or Mg concentration. Gel run under these conditions showed enhanced concentrations of slow-moving components, indicative of ligation of intermolecular associated DNA species. At a 10-fold higher level of ligase, ethanol appeared to unwind the duplex, confirming the results of Lee, Mizusawa, and Kakefuda [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2838-2842]. All of these anomalies occur under solvent conditions which are close to conditions which produce a heterogeneous dispersion of sedimenting species in ultracentrifugal experiments and compact rodlike structures, visualized by electron microscopy. The circular dichroism spectra at the onset of the formation of these structures show the characteristics of a chirally packed array of DNA duplexes. The reversal of the trend of the ethanol effect on linking number at higher enzyme and Mg(II) concentrations can be most easily explained by the promotion of the condensation phenomenon by either the ligase or a contaminating factor in the preparation. We suggest that the anomalies in the linking number and winding angle values are due to either ligation of chirally bent DNA species or a change in the helical period as the linear DNA adapts to the conformation required for collapse.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biotransformations of organosilicon compounds: enantioselective reduction of acyl silanes by means of baker's yeast

The enantioselective reduction of acyl silanes has been performed employing baker's yeast (BY) in fermenting conditions: a series of substrates of different structure was investigated, showing that the reactivity as well as the level of enentioselectivity depends on the steric bulk of the substituents on the acyl silane. The products α-hydroxy silanes were obtained with chemical and optical yields over 90% in the most favourable cases.     

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), and CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA.TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A.T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in two different structures are reported.     

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.