Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution.

We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of sigma= -0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA superhelix were analysed. Evaluation of the whole plasmid yielded average superhelix dimensions of 27 +/- 9 nm (outer superhelix diameter D), 107 +/- 51 nm (superhelix pitch P), and 54 +/-8 degrees (superhelix pitch angle alpha). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 +/- 3.3 nm, P = 42 +/- 13 nm and alpha= 63 +/- 20 degrees for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface.     

Time-Lapse Imaging of Conformational Changes in Supercoiled DNA by Scanning Force Microscopy

Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.     

Probing specific molecular conformations with the scanning force microscope. Complexes of plasmid DNA and anti-Z-DNA antibodies.

An anti-Z-DNA IgG antibody was used to probe for the left-handed Z-DNA conformation of a d(CG)11 insert in a negatively supercoiled plasmid DNA (pAN022). The complexes were spread on mica in the presence of a quaternary ammonium detergent benzyldimethylalkylammonium chloride and imaged with a scanning force microscope (SFM). The high affinity anti-Z-DNA antibody was retained even after restriction endonuclease cleavage of the DNA. The two arms in the product molecules had unequal lengths in conformity with the known location of the Z-DNA forming insert. Most complexes exhibited one IgG per DNA molecule. The bound antibodies were up to approximately 35 nm in diameter and extended approximately 2 nm from the mica surface. They were generally in a lateral orientation relative to the DNA, in accordance with prior chemical modification experimental data indicating a bipedal mode of binding for an anti-Z-DNA IgG. However, the SFM images also suggest that the DNA bends to accommodate the two Fab combining regions of the antibody. This study demonstrates the utility of the SFM for investigating conformation-dependent molecular recognition.     

Polylysine-coated mica can be used to observe systematic changes in the supercoiled DNA conformation by scanning force microscopy in solution

The conformations of supercoiled (sc) DNA and linear DNA bound to polylysine (PL)-coated mica were investigated by scanning force microscopy (SFM) in solution. From the polymer statistical analysis of linear DNA, we could distinguish between re-arrangements or trapping of the DNA on the surface. Conditions of re-arrangements to an almost equilibrated state can be achieved at appropriate PL surface concentrations. We could show that the ability of re-arrangements depends on the salt concentration of the adsorption/imaging buffer. Comparing the statistical analysis of the linear DNA with SFM images of scDNA suggested that irregular scDNA conformations are formed under conditions of trapping, whereas plectonemic structures are favoured under conditions of surface re-arrangements. Salt-dependent changes in the scDNA conformation over the range of 10–100 mM NaCl, as characterised by the parameters writhe and the superhelix radius r, are observable only under conditions that enable surface re-arrangements. The measured values of writhe suggest that the scDNA loses approximately one-half of the supercoils during the binding to the surface. At the same time r increases systematically with decreasing writhe, thus the scDNA topology remains determined by the constraints on supercoiling during the binding to PL-coated mica.     

Cell-surface receptors and proteins on platelet membranes imaged by scanning force microscopy using immunogold contrast enhancement.

High resolution scanning force microscope (SFM) images of fibrinogen-exposed platelet membranes are presented. Using ultrasharp carbon tips, we are able to obtain submolecular scale resolution of membrane surface features. Corroboration of SFM results is achieved using low voltage, high resolution scanning electron microscopy (LVHRSEM) to image the same protein molecule that is seen in the SFM. We obtain accurate height dimensions by SFM complemented by accurate lateral dimensions obtained by LVHRSEM. The use of 14- and 5-nm gold labels to identify specific membrane-bound biomolecules and to provide contrast enhancement with the SFM is explored as a useful adjunct to observation of unlabeled material. It is shown that the labels are useful for locating specific protein molecules on platelet membrane surfaces and for assessing the distribution of these molecules using the SFM. Fourteen nm labels are shown to be visible over the membrane corrugation, whereas 5-nm labels appear difficult to resolve using the present SFM instrumental configuration. When using the 5-nm labels, collateral use of LVHRSEM allows one to examine SFM images at submolecular resolution and associate function with the structures imaged after the SFM experiment is completed.     

Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips

In this work we have designed a simple system to investigate empirically the image contrast of tapping-mode atomic force microscopy (TMAFM). We modified the cantilever tips with protein molecules (bovine serum albumin or goat anti-biotin antibody) and used these protein-modified cantilevers to scan poly-L-lysine films and antibody layers deposited on mica in air under ambient conditions. We also investigated the effects of manipulating the setpoint voltage in this system. It was found that extra topographic features with a patchlike appearance were introduced into the TMAFM images of both the poly-L-lysine and antibody films when scanned with the protein-modified tips, even at initial preset setpoints, and were superimposed on the topography of the samples. The surface coverage of the patchlike features in the TMAFM images changes significantly with the setpoint voltage in a reversible and nonlinear manner. These are believed to arise from the surface indentation of the sample or from the structural deformation of the proteins at the tip induced in TMAFM imaging. Interestingly, it was observed in the experiment that no structural alteration or damage was discernible on the sample surface, even after continuous scanning with the protein-modified tips for a long period of time, with varying setpoint voltage. This study provides experimental evidence that cantilever tips modified with protein molecules or, under certain circumstances, even unmodified tips introduce extra topographical features (i.e., artifacts) and enhance the image contrast of TMAFM imaging of soft materials, which is dependent on their mechanical properties.     

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.     

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.     

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Sequence-dependent DNA curvature and flexibility from scanning force microscopy images

This paper reports a study of the sequence-dependent DNA curvature and flexibility based on scanning force microscopy (SFM) images. We used a palindromic dimer of a 1878-bp pBR322 fragment and collected a large pool of SFM images. The curvature of each imaged chain was measured in modulus and direction. It was found that the ensemble curvature modulus does not allow the separation of static and dynamic contributions to the curvature, whereas the curvature, when its direction in the two dimensions is taken into account, permits the direct separation of the intrinsic curvature contributions static and dynamic contributions. The palindromic symmetry also acted as an internal gauge of the validity of the SFM images statistical analysis. DNA static curvature resulted in good agreement with the predicted sequence-dependent intrinsic curvature. Furthermore, DNA sequence-dependent flexibility was found to correlate with the occurrence of A.T-rich dinucleotide steps along the chain and, in general, with the normalized basepair stacking energy distribution.     

S-DNA is oversupercoiled DNA with 1.94-2.19 A rise per base pair along duplex axis

Supercoiled pGEMEX DNA with length of 3993 nucleotides was immobilized on four substrates (freshly cleaved mica, standard amino mica, modified amino mica with increased and decreased surface charge density compared with standard amino mica) and it was visualized by atomic force microscopy (AFM) in air. Plectonomically supercoiled DNA molecules as well as single molecules with extremely high level of compaction (i.e. molecules with significantly higher superhelix density values on comparison with previously experimentally measured and theoretically investigated ones) were visualized on modified amino mica which was characterized by increased surface charge density. Distance between base pairs along duplex axis was determined by measurements of contour length of single oversupercoiled DNA molecules. Determined rise per base pair was varied from 1.94 to 2.19 A. These compressed supercoiled DNA molecules like a spring with decreased rise/base pair on comparison with well-known DNA forms were called new DNA form--S-DNA. A model of S-DNA was built. Formation of the S-DNA molecules was suggested to be an intermediate stage on the compaction of the single supercoiled DNA molecules up to the spheroids and minitoroids. Oversupercoiling and further compression of the supercoiled DNA molecules was shown to cause by high surface charge density of amino mica on which DNA molecules were immobilized.     

Atomic Force Microscopy Imaging of Double Stranded DNA and RNA

Abstract

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.     

Scanning force microscopy of DNA molecules elongated by convective fluid flow in an evaporating droplet.

Scanning force microscopy (SFM) was used to image intact, nearly fully elongated lambda bacteriophage DNA molecules, fixed onto freshly cleaved mica surfaces. Molecular elongation and fixation were accomplished using a newly characterized fixation technique, termed "fluid fixation." Here convective fluid flows generated within an evaporating droplet of DNA solution efficiently elongate DNA molecules for fixation onto suitably charged surfaces. SFM images of a very large bacteriophage genome, G, showed the presence of double-stranded bubbles. We speculate that these structures may contain putative replication forks. Overall, the experiments presented here demonstrate the viability of using fluid fixation for the preparation of DNA molecules for SFM imaging. The combination of largely automatable optically based techniques with the high-resolution SFM imaging presented here will likely produce a high-throughput system for detailed physical mapping of genomic DNA or clones.     

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.     

Atomic force microscopy of oriented linear DNA molecules labeled with 5nm gold spheres.

The atomic force microscope (AFM;1) can image DNA and RNA in air and under solutions at resolution comparable to that obtained by electron microscopy (EM) (2-7). We have developed a method for depositing and imaging linear DNA molecules to which 5nm gold spheres have been attached. The gold spheres facilitate orientation of the DNA molecules on the mica surface to which they are absorbed and are potentially useful as internal height standards and as high resolution gene or sequence specific tags. We show that by modulating their adhesion to the mica surface, the gold spheres can be moved with some degree of control with the scanning tip.     

Atomic force microscope measurements and manipulation of Langmuir-Blodgett films with modified tips.

A simple method for rendering atomic force microscope tips and cantilevers hydrophilic or hydrophobic through glow discharge in an appropriate gas atmosphere is introduced. Force curves at different humidities of these modified cantilevers were taken on freshly cleaved mica (hydrophilic surface) and on a monolayer of dipalmitoylphosphatidylethanolamine transferred onto mica (hydrophobic surface) to characterize the behavior of the cantilevers on hydrophilic and hydrophobic surfaces. Furthermore, Langmuir-Blodgett bilayers, with a dipalmitoylphosphatidylethanolamine bottom layer and a dipalmitoylphosphatidylcholine top layer, were imaged in the constant force mode in a multimode atomic force microscope in air under controlled humidity conditions. The friction and elasticity signal were recorded parallel to the topography. By varying the force exerted by the tip on the sample, different layers of the Langmuir-Blodgett system could be removed or flattened. Removal exposed underlying layers that exhibited a different friction and elasticity behavior. Furthermore, force scans with tips rendered hydrophobic were taken on the different layers of the sample to characterize the hydrophilic/hydrophobic nature of the layers. Only by combining the results obtained by the different methods can the structure of the lipid layer systems be identified.     

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy.

Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.     

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.     

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.