S-DNA, over-supercoiled DNA with a 1.94-to 2.19-Å rise per base pair

Supercoiled 3993-bp pGEMEX DNA immobilized on four substrates (freshly cleaved mica, standard amino mica, and modified amino mica with an increased or decreased surface charge density in comparison to standard amino mica) has been visualized by atomic force microscopy in the air. Plectonomically supercoiled DNA molecules, as well as single molecules with an extremely high compaction level (i.e., with a significantly higher superhelix density compared to those previously observed experimentally or estimated theoretically), have been visualized on modified amino mica with an increased surface charge density. The distance between nucleotide pairs along the duplex axis has been determined by measuring the contour length of individual oversupercoiled DNA molecules. The estimated rise per base pair varies from 1.94 to 2.19 Å. These supercoiled DNA molecules, which are compressed like a spring and have a decreased rise per base pair compared to previously known DNA forms are considered to be a new form of DNA, S-DNA. A model of S-DNA has been constructed. Molecules of S-DNA may be an intermediate in the course of the compaction of single supercoiled DNA molecules into spheroids and minitoroids. The DNA oversupercoiling, followed by the compression of the supercoiled molecules, has been shown to be accounted for by a high surface charge density of amino mica on which DNA molecules are immobilized.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second-and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second- and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

Compaction of single molecules of supercoiled DNA immobilized on amino mica: from duplex to minitoroidal and spheroidal conformations

Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica.     

Oversupercoiling and compactization of supercoiled DNA

Supercoiled DNA pGEMEX with length of 3993 nucleotides was immobilized on the different substrates (freshly cleaved mica, standard aminomica and modified aminomica) and visualized by atomic force microscopy. Plectonomically supercoiled DNA molecules as well as molecules with extremely high level of compactization (i.e. molecules with considerably higher supercoiled density values in comparing with experimentally measured and theoretically investigated ones) were visualized on modified aminomica. At the further increasing of the compactization level an axis length of oversupercoiled molecules was decreased from approximately 390 nm to approximately 140 nm and formation of minitoroids of approximately 50 nm diameter and molecules in sphere conformation were observed. Model of possible conformational transitions of supercoiled DNA was proposed basing on the analysis of captured AFM images at the increasing of supercoiling density.     

Amino-modified probes with immobilized linkers and proteins for atomic force microscopy

Functionalized by bovine serum albumin (BSA) probes for atomic force microscopy (AFM) which can be used for molecular recognition studies has been obtained. Modification and functionalization procedure of AFM probe includes three stages. First, amino probes were obtained by modification in vapors of amino silane derivative. Then surface amino groups of the amino probe interacted with homobifunctional amino reactive crosslinker. And finally, the probe with covalently attached crosslinker was functionalized by BSA molecules. Obtained AFM probes were characterized on the different stages of the modification by force measurements and the adhesion forces were determined. Process of modification was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and amino mica modified by crosslinker.     

Controlled immobilization of DNA molecules using chemical modification of mica surfaces for atomic force microscopy: characterization in air

Immobilization of biomolecules on surfaces while keeping the maximum conformational flexibility of the molecules is one of the most important techniques for atomic force microscopy imaging. We have developed two methods of controlling adsorption of DNA molecules on mica surfaces. The first method is the use of a mica surface modified with diluted 3-aminopropyltriethoxysilane (APS). Here we named this a "diluted APS-treated mica (AP-mica)" technique. The second method is the use of a mica surface modified with mixed self-assembled monolayers of organosilanes. In both of the techniques, the number of DNA molecules immobilized on a mica surface was controlled. Further, a conformational change of circular DNA, from a supercoiled to a relaxed form was observed for the molecules immobilized on a diluted AP-mica surface, when 254-nm UV light was irradiated. This observation demonstrated that flexibility of circular DNA molecules was kept on a diluted AP-mica surface.     

Functionalization of amino-modified probes for atomic force microscopy

The probes for atomic force microscopy (AFM) functionalized with bovine serum albumin (BSA) were obtained; they can be used for molecular recognition studies. The procedure of modification and functionalization of the AFM probe included three stages. First, amino probes were obtained by modification in vapors of an amino silane derivative. Then, a covalent bond was formed between the surface amino groups of the probe and a homobifunctional aminoreactive crosslinker. Finally, the probe with a covalently attached crosslinker was functionalized with BSA molecules. The AFM probes were characterized by force measurements at different stages of the modification; the adhesion force and the work of adhesion force were determined. The modification process was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and on amino mica modified with a crosslinker.     

Functionalization of aminomodified probes for atomic force microscopy

Probes for atomic force microscopy functionalized by bovine serum albumin were obtained, which may be used for molecular recognition studies. The procedure of the modification and functionalization of probes includes three stages. First, amino probes are obtained by modification in vapors of amino silane derivative. Then a homobifunctional amino reactive cross-linker is covalently linked to surface amino groups of the amino probe. And finally, the probe with the covalently attached cross-linker is functionalized by bovine serum albumin molecules. The probes obtained were characterized at different stages of the modification by atomic force microscopy: the adhesion force and the work of adhesion force were determined from histograms. The modification of probe surface was confirmed by visualization of bovine serum albumin and supercoiled pGEMEX DNA molecules immobilized on the amino mica and amino mica modified by cross-linker.     

Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate.

A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules.     

Study of microorganism genome DNA by atomic force microscopy

The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.     

The visualization of amplicons after polymerase chain reaction

Linear DNA molecules amplified by the polymerase chain reaction were visualized by atomic force microscopy. The measured contour length of the PCR product of 1414 bp sequence was 435 +/- 15 nm. Considering that the calculated value of the distance between the nucleotides along the duplex axis is 0.31 nm, it was assumed that linear DNA molecules on the surface of mica, which serve as a support in the atomic force microscopy method, are in the A form. The influence of surface properties of the mica and the sample drying procedure on the conformation of adsorbed DNA molecules is discussed. Possible reasons for the Gaussian distribution of the contour length of the synthesized amplicon are considered.     

Visualization of the cruciform structure of superhelical DNA by use of atomic force microscopy

Atomic force microscopy was used to visualize the cruciform structure in supercoiled plasmid pUC8 DNA immobilized on aminomodified mica. The cruciform hairpin was 14 base pairs in size, as determined from atomic force microscopy images of pUC8 DNA in air. Molecular modeling confirmed that the cruciform structure is formed by hairpins with self-complementary homopyrimidine-homopurine sequences (dT)8(dA)6 and a loop 4 nucleotides long.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.     

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.     

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.