Force Microscopy In Solution Research Articles

DNA-intercalator interactions: structural and physical analysis using atomic force microscopy in solution

DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since attracted considerable research scrutiny. DNA intercalators (molecules that intercalate between DNA base pairs) have attracted particular attention due to their antitumoral activity and because they are widely used to fluorescently label DNA. The interaction between double-stranded DNA and bis-intercalators, such as the fluorescent dye YOYO-1 (dimer of oxazole yellow), has been widely studied. In the literature, there are contradicting data regarding the structure and the rigidity of the complex DNA–YOYO-1. Here, we address this problem using high-resolution atomic force microscopy in solution. We directly measured important structural parameters, such as the helical pitch and the elongation of the complex. By measuring intercalator-induced DNA elongation at different YOYO-1 concentrations, we determined the binding constant. We showed that intercalation induces distinct changes in the molecular elasticity compared to the free double stranded DNA. We found a decrease of the persistence length of DNA with increasing amount of bound YOYO-1, which contrasts with some previous assumptions. This study helps in understanding the physicochemical properties of bis-intercalators as well as the mechanism by which they interact with DNA and this technique can ultimately be applied to a large range of other DNA binding molecules such as anti-cancer or anti-viral drugs.

Conformational analysis and estimation of the persistence length of DNA using atomic force microscopy in solution

The worm-like chain model describes the mechanical properties of semi-flexible polymers by introducing a certain correlation length along the contour. This correlation length is called the persistence length. Using atomic force microscopy in solution, we performed measurements of the persistence length of DNA molecules. We found good agreement between the theoretical model and experimental data. However, the measured persistence length values in solution differ from those found by several authors using the same technique but in dry air. In order to determine the contribution of the electrostatic persistence length to the total persistence length, we varied the salt concentration. We found a large discrepancy between the Odijk, Skolnick and Fixman theory and our measurements. The effect of divalent ions and the omission in the theory of the dependence of non-electrostatic persistence length on salt concentration are qualitatively invoked.

Assessing mutant huntingtin fragment and polyglutamine aggregation by atomic force microscopy

Huntington disease (HD), a neurodegenerative disorder, is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats located near the N-terminus of the huntingtin (htt) protein. The expansion of the polyQ domain results in the ordered assembly of htt fragments into fibrillar aggregates that are the main constituents of inclusion bodies, which are a hallmark of the disease. This paper describes protocols for studying the aggregation of mutant htt fragments and synthetic polyQ peptides with atomic force microscopy (AFM). Ex situ AFM is used to characterize aggregate formation in protein incubation as a function of time. Methods to quickly and unambiguously distinguish specific aggregate species from complex, heterogeneous aggregation reactions based on simple morphological features are presented. Finally, the application of time lapse atomic force microscopy in solution is presented for studying synthetic model polyQ peptides, which allows for tracking the formation and fate of individual aggregates on surfaces over time. This ability allows for dynamic studies of the aggregation process and direct observation of the interplay between different types of aggregates.

Visualization of Hydrophobic Polyelectrolytes Using Atomic Force Microscopy in Solution

We have used atomic force microscopy to study the morphology of hydrophobic polyelectrolytes adsorbed on surfaces. The polyelectrolytes consisted of polystyrene sulfonate (PSS) chains made with three charge densities: 32%, 67%, and 92%. They were adsorbed on two types of surfaces: mica, and phospholipid bilayers made of mixed neutral and cationic lipids. We show that the chains with a low charge density (32%) are collapsed in spherical globules while highly charged chains (67% and 92%) are fully extended. End-to-end distances and contour lengths of the extended chains were measured. Statistical analysis shows that the persistence length of these chains depends on the surface where they adsorb. On lipid bilayers, highly ordered monolayers are formed upon increase of the proportion of cationic phospholipids. These results show that highly charged PSS chains behave in a similar manner than the stiffer, hydrophilic DNA when adsorbed on surfaces. It could lead to the design of new types of nanostructured surfaces using polyelectrolyte molecules synthesized with specific properties.

Inter-Oligomer Interactions of the Human Prion Protein Are Modulated by the Polymorphism at Codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (βO) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the βO N-terminal segments, showing that it is the core region of PrP (residues 127–228) that is involved in βO formation. Atomic force microscopy revealed distinctive 1.8×15×15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for βO with valine at position 129. This result provides evidence for conformational differences between the βO allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.

Adhesion and Stable Low Friction Provided by a Subnanometer-Thick Monolayer of a Natural Polysaccharide

Using a surface forces apparatus, we have investigated the adhesive and lubrication forces of mica surfaces separated by a molecularly thin, subnanometer film of a high-molecular-weight (2.3 MDa) anionic polysaccharide from the algae Porphyridium sp. adsorbed from aqueous solution. The adhesion and friction forces of the confined biopolymer were monitored as a function of time, shearing distance, and driving velocity under a large range of compressive loads (pressures). Although the thickness of the dilute polysaccharide was <1 nm, the friction was low (coefficient of friction = 0.015), and no wear was ever observed even at a pressure of 110 atm over 3 decades of velocity, so long as the shearing distances were less than twice the contact diameter. Atomic force microscopy in solution shows that the biopolymer is able to adsorb to the mica surface but remains mobile and easily dragged upon shearing. The adhesion (adsorption) of this polysaccharide even to negatively charged surfaces, its stable low friction, its robustness (high-load carrying capacity and good wear protection), and the weak (logarithmic) dependence of the friction force on the sliding velocity make this class of polyelectrolytes excellent candidates for use in water-based lubricant fluids and as potential additives to synovial fluid in joints and other biolubricating fluids. The physical reasons for the remarkable tribological properties of the ultrathin polysaccharide monolayer are discussed and appear to be quite different from those of other polyelectrolytes and proteins that act as thick "polymer brush" layers.

Atomic force microscopy of DNA in solution and DNA modelling show that structural properties specify the eukaryotic replication initiation site

The replication origins (ORIs) of Schizosaccharomyces pombe, like those in most eukaryotes, are long chromosomal regions localized within A+T-rich domains. Although there is no consensus sequence, the interacting proteins are strongly conserved, suggesting that DNA structure is important for ORI function. We used atomic force microscopy in solution and DNA modelling to study the structural properties of the Spars1 origin. We show that this segment is the least stable of the surrounding DNA (9 kb), and contains regions of intrinsically bent elements (strongly curved and inherently supercoiled DNAs). The pORC-binding site co-maps with a superhelical DNA region, where the spatial arrangement of adenine/thymine stretches may provide the binding substrate. The replication initiation site (RIP) is located within a strongly curved DNA region. On pORC unwinding, this site shifts towards the apex of the curvature, thus potentiating DNA melting there. Our model is entirely consistent with the sequence variability, large size and A+T-richness of ORIs, and also accounts for the multistep nature of the initiation process, the specificity of pORC-binding site(s), and the specific location of RIP. We show that the particular DNA features and dynamic properties identified in Spars1 are present in other eukaryotic origins.

Dissolution of Brucite on the (001) Surface at Neutral pH: in situ Atomic Force Microscopy Observations

AbstractThe dissolution of brucke, Mg(OH)2, on the (001) surface was investigated using in situ atomic force microscopy in solutions at near-neutral pH. Dissolution proceeded by the formation of crystallographically oriented triangular etch pits with monolayer step and expansion of the pits. The sides of the triangle are parallel to the [100], [110] and [010] directions of the brucite structure, and the orientation of lines from the center of the triangle to the three apices are along the [210], [1¯10]$[\bar 110]$ and [1¯2¯0]$[\bar 1\bar 20]$ directions. This orientation may produce pit edges where OH groups coordinate to two Mg2+. Although triangular etch pits with monolayer depth formed mostly at random on the (001) surface, concentric pits penetrating several layers were also observed. Etch pits with spiral steps were rarely observed. Coalescence of the pits resulted in stranded terraces that diminished in size rapidly and formed a rounded irregular form. The step-retreat velocity around the triangular pit is 0.015–0.04 nm/s at pH 5–8. The retreat velocity around the stranded terraces was about three times more rapid than that around the triangular etch pits.

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.

Visualizing filamentous actin on lipid bilayers by atomic force microscopy in solution.

The surface structure of actin filaments (F-actin) was visualized at high resolution, by atomic force microscopy (AFM) in aqueous solution, in large paracrystals prepared on positively charged lipid monolayers. The increased stability of these closely packed specimens allowed us to show that both the long pitch (38 nm) and the monomer (5.8 nm) can be directly resolved by AFM in the contact mode. The right-handed helical surface, distinguishable in high resolution images, was compared with reconstructed models based on electron microscopy. The height of the rafts, a measure of the actin filament diameter, was 10 +/- 1 nm, whereas the smaller inter-filament distance, 8 +/- 1 nm, was consistent with interdigitation of the filaments. The 10 +/- 1 nm F-actin diameter is in good agreement with the results of fibre X-ray diffraction. As such specimens are relatively easy to prepare without specialized equipment, this method may allow the study of the thin filaments in which F-actin-associated proteins are also present.

A piezotube scanner for atomic force microscopy in solution

A novel piezotube scanner used in the atomic force microscope (AFM) is described. Unlike other designs where the electrodes on the outside are subjected to high voltage, in our design all electrodes are placed on the inside of the tube scanner, and the outside electrode is simply grounded. With this design and a proper choice of piezomaterials, the scan range of a 1.75 in. tube can be as large as 15 μm when used with NanoScope III, and is not vulnerable to leaking solutions, which often cause severe damage to both the piezotube and the high-voltage driver with the other designs. Therefore, this new design will be valuable for biological AFM where imaging in solution is often required, and is simple enough to be fitted to an existing AFM.