Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

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.     

Influence of DNA condensation state on transfection efficiency in DNA/polymer complexes: an AFM and DLS comparative study

Atomic force microscopy (AFM) is used to describe the formation process of polymer/DNA complexes. Two main objectives of this research are presented. The first one is to apply AFM as an effective tool to analyse DNA molecules and different polycation/DNA complexes in order to evaluate their degree of condensation (size and shape). The other one is to search for a relationship between the condensation state of DNA and its transfection efficiency. In this study, linear methacrylate based polymers and globular SuperFect polymers are used in order to induce DNA condensation. Ternary complexes, composed of methacrylate based polymers and polyethylene glycol (PEG)-based copolymers, are also investigated. AFM allows us to confirm good condensation conditions and relate them (or not) to transfection efficiencies. These AFM results (obtained after drying in air) are compared with measurements deduced from Dynamic Light Scattering (DLS) experiments performed in water. This comparison allowed us to identify the structural modifications resulting from deposition on the mica surface.     

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.     

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg²⁺-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg²⁺ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.     

Circular dichroism studies on DNA condensed in NaCl cetyltrimethyl ammonium bromide solutions.

It is shown by means of circular dichroism studies of variously condensed forms of DNA that the specific supramolecular structure of DNA determines the type of CD spectra. DNA, condensed (crystallized) slowly in the presence of cetyltrimethyl ammonium bromide yields a spectrum very similar to that of DNA in solution in the B-form. The condensates appear in the phase-contrast microscope as spherulitic crystallites. Rapidly condensed DNA in the presence of cetyltrimethyl ammonium bromide shows a spectrum of the psi-type with large negative ellipticites. The influence of condensation velocity upon the supramolecular structure of DNA gives evidence that the various condensation forms of DNA are not thermodynamical equilibrium conformations.     

Nanoscale time-lapse AFM imaging in solution for DNA aggregation

Revealing the behavior of biofunctional molecules (i.e., nucleic acids, nucleic acid binding reagents, enzymatic proteins, etc.) by monitoring them in solution is important for understanding the nanoscale dynamism of their interactions. Atomic force microscope (AFM) imaging with a dynamic force mode (DFM, i.e., tapping mode) in aqueous solution, has many advantages for the imaging of DNA morphological change at a single molecule scale. Hoechst 33258 (H33258) induces DNA condensation in the presence of its excess concentration. To have a better understanding of the condensation process of DNA with excess H33258, we tried to find the optimum conditions for carrying out time-lapse AFM imaging in aqueous solution. To immobilize DNA on the substrate surface, the mica was modified with the various concentrations of 3-aminopropyltriethoxysilane (APTES) solution. We observed that DNA was minimally immobilized on 0.002% APTES-modified mica surface. Then, we determined that the movement of DNA on the mica surface could be observed in the presence of 500 mM NaCl in 10 mM PBS (pH 7.0). Moreover, after the injection of 5 μM H33258, the partial condensation of DNA was observed.     

The structure of the complexes of DNA with chromosomal protein HMGB1 and histone H1 in the presence of manganese ions. I. Circular dicroism spectroscopy

The mechanisms of interaction of the non-histone chromosomal protein HMGB1 and linker histone H1 with DNA have been studied using circular dichroism and absorption spectroscopy. Both of the proteins are located in the inter-nucleosomal regions of chromatin. It was demonstrated that properties of the DNA-protein complexes depend on the protein content and can not be considered as a simple summing up of the effects of individual protein components. Interaction of HMGB1 and H1 proteins is shown to be co-operative rather than competitive. Lysine-rich histone H1 facilitates the binding of the HMGB1 with DNA by screening the negatively charged groups of the sugar-phosphate backbone of DNA and dicarboxylic amino-acid residues in the C-terminal domain of the HMGB1 protein. The observed joint action of the and H1 proteins stimulates DNA condensation with formation of the anisotropic DNA-protein complexes with typical psi-type CD spectra. Structural organization of the complexes depends not only on the DNA-protein interactions, but also on the interaction between HMGB1 and H1 protein molecules bound to DNA. Manganese ions significantly modify the character of interactions between the components in the triple DNA-HMGB1-H1 complex. Binding of Mn2+ ions causes the weakening of the DNA-protein interactions and strengthening the protein-protein interactions, which promote DNA condensation and formation of large DNA-protein particles in solution.     

The structural transition of DNA-Tris(1,10-phenanthroline) cobalt(III) complexes in ethanol-water solution

The interaction of DNA with Tris(1,10-phenanthroline) cobalt(III) was studied by means of atomic force microscopy. Changes in the morphologies of DNA complex in the presence of ethanol may well indicate the crucial role of electrostatic force in causing DNA condensation. With the increase of the concentration of ethanol, electrostatic interaction is enhanced corresponding to a lower dielectric constant. Counterions condense along the sugar phosphate backbone of DNA when epsilon is lowered and the phosphate charge density can thus be neutralized to the level of DNA condensation. Electroanalytical measurement of DNA condensed with Co(phen)(3)(3+) in ethanol solution indicated that intercalating reaction remains existing. According to both the microscopic and spectroscopic results, it can be found that no secondary structure transition occurs upon DNA condensing. B-A conformation transition takes place at more than 60% ethanol solution.     

Atomic force microscopy analysis of intermediates in cobalt hexammine-induced DNA condensation

The packaging pathway of cobalt hexammine-induced DNA condensation on the surface of mica was examined by varying the concentration of Co(NH3)6(3+) in a dilute DNA solution and visualizing the condensates by atomic force microscopy (AFM). Images reveal that cobalt hexammine-induced DNA condensation on mica involves well-defined structures. At 30 microM Co(NH3)6(3+), prolate ellipsoid condensates composed of relatively shorter rods with linkages between them are formed. At 80 microM Co(NH3)6(3+), the condensed features include toroids with average diameter of approximately 240 nm as well as U-shaped and rod-like condensates with nodular appearances. The results imply that the condensates, whether toroids, U-shaped or rod-like structures have similar intermediate state which includes relatively shorter rod-like segments. The average size of the condensed toroids after incubated at room temperature for 5 h (approximately 240 nm) is much larger than that incubated for 0.5 h (approximately 100 nm). The results indicate that the condensation of DNA by Co(NH3)6(3+) is a kinetic-controlled process.     

Secondary structure of condensed DNA. wide-angle, small-angle x-ray scattering and circular dichroism

Ethanol precipitated DNA shows a CD spectrum of the +psi-type which is similar to that of DNA in the A-form. DNA condensed with cetyl-trimethylammonium-bromide shows, depending on the condensation velocity, a CD spectrum of the -psi-type, or a CD spectrum only slightly modified from that of DNA in solution. The first spectrum is similar to that of DNA in the C-form, and the second one, to that of DNA in the B-form. Using large-angle X-ray scattering of the three DNA condensates and comparing them with the scattering curves calculated from the atom coordinates for the A-, B-, and C-form of DNA it is shown that the secondary structure of the DNA belongs in all three cases to the B-family. It follows from this result that the secondary structure of DNA alone does not determine the type of CD spectrum. The CD spectrum of condensed DNA is essentially determined by the supramolecular structures of the partially crystalline DNA condensates. These supramolecular structures can be demonstrated by the small-angle X-ray diagrams. The condensation of DNA by ethanol and cetyl-trimethylammonium-bromide proceeds in the form of a partial crystallization of the DNA.     

Daunomycin inverts the long-range chirality of DNA condensed states

The effect of daunomycin upon DNA condensed states induced by poly(ethylene glycol) (PEG) was studied by circular dichroism (CD) and circular intensity differential scattering (CIDS). The CD spectra of these aggregates showed psi-type anomalies and intensities 10-100 times greater than those obtained with the dispersed DNA solutions in the absence of PEG. Increasing concentrations of daunomycin, added to the DNA solution prior to its aggregation, led, in the presence of PEG, to CD and CIDS signals which gradually decreased in magnitude and eventually inverted sign. The coincidence of the transition point of both signals and a careful characterization of the CD spectrum at the transition point clearly indicated that the inversion observed corresponds to an inversion of the handedness of the aggregates. The latter result suggests that the structure of the aggregates at the inversion point should resemble that of a nematic liquid-crystalline structure. The characteristic B-DNA spectrum obtained in this case further suggests that the packing process does not affect the secondary structure of the DNA molecules and that small changes in their local structure can induce dramatic changes in their long-range tertiary packing. The results obtained in this study represent a confirmation of a recent theory of psi-type CD in which the anomalous signals are interpreted as a manifestation of the long-range chirality of the aggregates.     

Origin of enhanced VCD in amyloid fibril spectra: Effect of deuteriation and pH

Supramolecular chirality of amyloid fibrils, protein aggregates related to many neurodegenerative diseases, is a remarkable property associated with fibril structure and polymorphism. Since its discovery almost 10 years ago there is still little understanding of this phenomenon, including the cause of the highly enhanced vibrational circular dichroism (VCD) intensity arising from fibril supramolecular chirality. In this study, VCD spectra, enhanced by filament supramolecular chirality, are presented for lysozyme and insulin fibrils above and below pH 2 and after deuterium exchange, above and below pD 2. Supramolecular chirality (observed by VCD) and fibril morphology (documented by atomic force microscopy) are not affected by protein deuteriation. In D₂O the fibril VCD sign pattern changes to fewer bands, with implications for the amide I/II origin of enhanced VCD intensity. Separation of amide I and II signals will facilitate calculations of enhanced VCD spectra of amyloid fibrils and enable a better understanding of the origin of the VCD sign pattern.     

AFM studies of DNA structures on mica in the presence of alkaline earth metal ions

As counterions of DNA on mica, Mg(2+), Ca(2+), Sr(2+) and Ba(2+) were used for clarifying whether DNA molecules equilibrate or are trapped on mica surface. End to end distance and contour lengths were determined from statistical analysis of AFM data. It was revealed that DNA molecules can equilibrate on mica when Mg(2+), Ca(2+) and Sr(2+) are counterions. When Ba(2+) is present, significantly crossovered DNA molecules indicate that it is most difficult for DNA to equilibrate on mica and the trapping degree is different under different preparation conditions. In the presence of ethanol, using AFM we have also observed the dependence of B-A conformational transition on counterion identities. The four alkaline earth metal ions cause the B-A transition in different degrees, in which Sr(2+) induces the greatest structural transition.     

AFM for analysis of structure and dynamics of DNA and protein-DNA complexes

This paper describes protocols for studies of structure and dynamics of DNA and protein-DNA complexes with atomic force microscopy (AFM) utilizing the surface chemistry approach. The necessary specifics for the preparation of functionalized surfaces and AFM probes with the use of silanes and silatranes, including the protocols for synthesis of silatranes are provided. The methodology of studies of local and global conformations DNA with the major focus on the time-lapse imaging of DNA in aqueous solutions is illustrated by the study of dynamics of Holliday junctions including branch migration. The analysis of nucleosome dynamics is selected as an example to illustrate the application of the time-lapse AFM to studies of dynamics of protein-DNA complexes. The force spectroscopy is the modality of AFM with a great importance to various fields of biomedical studies. The AFM force spectroscopy approach for studies of specific protein-DNA complexes is illustrated by the data on analysis of dynamics of synaptic SfiI-DNA complexes. When necessary, additional specifics are added to the corresponding example.     

Simulations of oligopeptide vibrational CD: effects of isotopic labeling

Simulated ir absorption and vibrational CD (VCD) spectra of four alanine-based octapeptides, each having its main chain constrained to a different secondary structure conformation, were analyzed and compared with experimental results for several different peptides. The octapeptide simulations were based on transfer of property tensors from a series of ab initio calculations for a short L-alanine based segment containing 3 peptide bonds with relative straight phi, psi angles fixed to those appropriate for alpha-helix, 3(10)-helix, ProII-like helix, and beta-sheet-like strand. The tripeptide force field (FF) and atomic polar tensors were obtained with density functional theory techniques at the BPW91/6-31G** level and the atomic axial tensor at the mixed BPW91/6-31G**/HF/6-31G level. Allowing for frequency correction due to the FF limitations, the octapeptide results obtained are qualitatively consistent with experimental observations for ir and VCD spectra of polypeptides and oligopeptides in established conformations. In all cases, the correct VCD sign patterns for the amide I and II bands were predicted, but the intensities did have some variation from the experimental patterns. Predicted VCD changes upon deuteration of either the peptide or side-chains as well as for (13)C isotopic labeling of the amide C=O at specific sites in the peptide chain were computed for analysis of experimental observations. A combination of theoretical modeling with experimental data for labeled compounds leads both to enhanced resolution of component transitions and added conformational applicability of the VCD spectra.     

Direct measurement of DNA by means of AFM.

We have demonstrated that the electrostatic stretch-and-positioning method is useful for the analysis of a long DNA molecule by means of atomic force microscopy (AFM). DNA molecules were stretched parallel to the field line, and immobilized onto the aluminum electrodes patterned on a glass plate. Through AFM observation, we confirmed the immobilization of individual DNA molecules, not aggregate.     

IR (vibrational) CD of peptide beta-turns: a theoretical and experimental study of cyclo-(-Gly-Pro-Gly-D-Ala-Pro-).

IR vibrational CD (VCD) has been observed for the cyclic pentapeptide cyclo-(-Gly-Pro-Gly-D-Ala-Pro-) in solution in CDBr3. The observed VCD spectra do not resemble the VCD features of any of the previously reported peptide secondary structures, such as alpha-helical, "random coil," or sheet structures, and might be due to the beta-turn contained in this molecule. To shed light onto the origin of the observed spectra, VCD intensity calculations, based on the solution and solid-state structures of cyclo-(-Gly-Pro-Gly-D-Ala-Pro-), have been carried out. In addition, calculated VCD data for pure beta-turns are discussed.