High-resolution atomic force microscopy of DNA

A method using high resolution atomic force microscopy for imaging DNA has been elaborated. Using super-sharp probes and modified graphite as support for molecule adsorption, DNA molecule images were obtained whose resolution made possible the observation of their fine structure with repeated helical motifs. The method can be used to visualize individual spread molecules of single-stranded DNA.     

Imaging the RecA-DNA complex by atomic force microscopy.

Atomic force microscopy (AFM) was applied to study the RecA protein and its complexes with DNA in air and in aqueous solution. RecA and DNA were reacted under several conditions, and deposited onto a mica substrate pre-treated in various ways. We found that the structure of the RecA and RecA-DNA complexes, especially the height of the molecules, was affected by the sample preparation method such as gel filtration, and environment during imaging.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Applications for atomic force microscopy of DNA.

Tapping mode atomic force microscopy (AFM) of DNA in propanol, dry helium, and aqueous buffer each have specific applications. Resolution is best in propanol, which precipitates and immobilizes the DNA and provides a fluid imaging environment where adhesive forces are minimized. Resolution on exceptional images of DNA appears to be approximately 2 nm, sufficient to see helix turns in detail, but the smallest substructures typically seen on DNA in propanol are approximately 6-10 nm in size. Tapping AFM in dry helium provides a convenient way of imaging such things as conformations of DNA molecules and positions of proteins on DNA. Images of single-stranded DNA and RecA-DNA complexes are presented. In aqueous buffer DNA molecules as small as 300 bp have been imaged even when in motion. Images are presented of the changes in shape and position of circular plasmid DNA molecules.     

Dynamics of synaptic SfiI-DNA complex: single-molecule fluorescence analysis

A single-molecule analysis was applied to study the dynamics of synaptic and presynaptic DNA-protein complexes (binding of two DNA and one DNA duplex, respectively). In the approach used in this study, the protein was tethered to a surface, allowing a freely diffusing fluorescently labeled DNA to bind to the protein, thus forming a presynaptic complex. The duration of fluorescence burst is the measure of the characteristic lifetime of the complex. To study the formation of the synaptic complex, the two SfiI-bound duplexes with the labeled donor and acceptor were used. The synaptic complex formation by these duplexes was detected by the fluorescence resonance energy transfer approach. The duration of the fluorescence resonance energy transfer burst is the measure of the characteristic lifetime of the synaptic complex. We showed that both synaptic and presynaptic complexes have characteristic dissociation times in the range of milliseconds, with the synaptic SfiI-DNA complex having the shorter dissociation time. Comparison of the off-rate data for the synaptic complex with the rate of DNA cleavage led to the hypothesis that the complex is very dynamic, so the formation of an enzymatically active synaptic complex is a rather rare event in these series of conformational transitions.     

Two-component atomic force microscopy recognition imaging of complex samples

Biological complexes are typically multisubunit in nature and the processes in which they participate often involve protein compositional changes in themselves and/or their target substrates. Being able to identify more than one type of protein in complex samples and to track compositional changes during processes would thus be very useful. Toward this goal, we describe here a single-molecule technique that can simultaneously identify two types of proteins in compositionally complex samples. It is an adaptation of the recently developed atomic force microscopy (AFM) recognition imaging technique but involves the tethering of two different types of antibodies to the AFM tip and sequential blocking with appropriate antigenic peptides to distinguish the recognition from each antibody. The approach is shown to be capable of simultaneously identifying in a single AFM image two specific components, BRG1 and beta-actin, of the human Swi-Snf ATP-dependent nucleosome remodeling complex and two types of histones, H2A and H3, in chromatin samples.     

Graphical method for force analysis: macromolecular mechanics with atomic force microscopy

We present a graphical method for a unifying, quantitative analysis of molecular bonding-force measurements by atomic force microscopy (AFM). The method is applied to interpreting a range of phenomena commonly observed in the experimental AFM measurements of noncovalent, weak bonds between biological macromolecules. The analysis suggests an energy landscape underlying the intermolecular force and demonstrates that many observations, such as "snaps-on," "jumps-off," and hysteresis loops, are different manifestations of a double-well energy landscape. The analysis gives concrete definitions for the operationally defined "attractive" and "adhesive" forces in terms of molecular parameters. It is shown that these operationally defined quantities are usually functions of the experimental setup, such as the stiffness of the force probe and the rate of its movement. The analysis reveals a mechanical instability due to the multistate nature of molecular interactions and provides new insight into macromolecular viscosity. The graphical method can equally be applied to a quantitative analysis of multiple unfolding of subunits of the giant muscle protein titin under AFM.     

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.     

Mechanical force analysis of peptide interactions using atomic force microscopy

Some peptides have previously been reported to bind low molecular weight chemicals. One such peptide with the amino acid sequence His-Ala-Ser-Tyr-Ser was selectively screened from a phage library and bound to a cationic porphyrin, 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphine (TMpyP), with a binding constant of 10(5) M(-1) (J. Kawakami, T. Kitano, and N. Sugimoto, Chemical Communications, 1999, pp. 1765-1766). The proposed binding was due to pi-electron stacking from two aromatic amino acids of histidine and tyrosine. In this study, the weak interactions between TMpyP and the peptide were further investigated by force curve analysis using atomic force microscopy (AFM). The mechanical force required to unbind the peptide-porphyrin complex was measured by vertical movement of the AFM tip. Peptide self-assembled monolayers were formed on both a gold-coated mica substrate and a gold-coated AFM tip. The TMpyPs could bind between the two peptide layers when the peptide-immobilized AFM tip contacted the peptide-immobilized substrate in solution containing TMpyP. In the retracting process a force that ruptured the interaction between TMpyPs and peptides was observed. The unbinding force values correlated to the concentration of TMpyP. A detection limit of 100 ng/mL porphyrin was obtained for the force measurement, and was similar to surface plasmon resonance sensor detection limits. Furthermore, we calculated the product of the observed force and the length of the molecular elongation to determine the work required to unbind the complexes. The obtained values of unbinding work were in a reasonable range compared to the binding energy of porphyrin-peptide.     

Stretched DNA structures observed with atomic force microscopy.

Double-stranded DNA molecules are occasionally found that appear to be straightened and stretched in atomic force microscope (AFM) images. Usually pBS+ plasmid and lambda DNA show relaxed structures with bends and kinks along the strands and have measured contour lengths consistent to about 5-7%; they also appear not to cross over each other, except in very high concentrations. The anomalous molecules observed here, compared with the majority of molecules in the preparation, show contour lengths increased by as much as 80% and have measured heights of about half that of normal relaxed DNA. Some molecules also appear to be in transition between stretched and relaxed forms. These observations are consistent with an uncoiling of the DNA helix without breakage of the covalent bonds in the deoxyribose-phosphate backbone.     

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.     

Quantum dot binding to DNA: Single-molecule imaging with atomic force microscopy

The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309     

Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partially homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation. Three-way junctions were obtained by annealing one strand of a fragment from a parental plasmid with one strand of an inverted repeat-containing fragment, purified from gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (AFM in situ). Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like. Non-flatness of the junction should be taken into account in analysis of the AFM data.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

H-NS mediated compaction of DNA visualised by atomic force microscopy

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS–DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.     

Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy

Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed.