Atomic force microscopy of DNA molecules stretched by spin-coating technique

We have developed an effective approach to stretching DNA molecules with the flow of fluid generated by spin coating. Well-stretched A DNA molecules were observed using atomic force microscopy. Substrate properties sensitively affected the stretching behavior of DNA. Our experimental findings revealed that a mica surface treated with crystal violet, a cationic dye molecule, is suitable to the spin-coating procedure for stretching DNA. Moreover, compared with relaxed DNA, we observed reduced height of the stretched DNA, which was attributed mainly to elongation force applied to the DNA molecules from the fluid flow and strong adhesion force between DNA and the substrate. This simple and effective method for preparing stretched DNA could be useful in physically mapping genomic DNA in a high throughput.     

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 microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.     

Atomic force microscopy of single- and double-stranded DNA.

A method has been developed for imaging single-stranded DNA with the atomic force microscope (AFM). phi X174 single-stranded DNA in formaldehyde on mica can be imaged in the AFM under propanol or butanol or in air. Measured lengths of most molecules are on the order of 1 mu, although occasionally more extended molecules with lengths of 1.7 to 1.9 mu are seen. Single-stranded DNA in the AFM generally appears lumpier than double-stranded DNA, even when extended. Images of double-stranded lambda DNA in the AFM show more sharp kinks and bends than are typically observed in the electron microscope. Dense, aggregated fields of double-stranded plasmids can be converted by gentle rinsing with hot water to well spread fields.     

Atomic force microscope investigation of large-circle DNA molecules

A circular bacterial artificial chromosome of 148.9kbp on human chromosome 3 has been extended and fixed on bare mica substrates using a developed fluid capillary flow method in evaporating liquid drops. Extended circular DNA molecules were imaged with an atomic force microscope (AFM) under ambient conditions. The measured total lengths of the whole DNA molecules were in agreement with sequencing analysis data with an error range of +/-3.6%. This work is important groundwork for probing single nucleotide polymorphisms in the human genome, mapping genomic DNA, manipulating biomolecular nanotechnology, and studying the interaction of DNA-protein complexes investigated by AFM.     

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L_W = 174 nm and a number average contour length of L_N = 132 nm (L_W/L_N = 1.32). A proportion of the pectin molecules (30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L_W = 137 nm, L_N = 108 nm with L_W/L_N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.     

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.     

Atomic force microscopy: a forceful way with single molecules

The atomic force microscope (AFM) now routinely provides images that reveal subnanometer surface structures of biomolecules. The sensitivity and precision of AFM provide new opportunities for studying the mechanical properties of biomolecules and their interactions in their native environment.     

Circular DNA molecules imaged in air by scanning force microscopy.

Routine and reproducible imaging of DNA molecules in air with the scanning force microscope (SFM) has been accomplished. Circular molecules of plasmid DNA were deposited onto red mica and imaged under various relative humidities. In related experiments, the first images of the Escherichia coli RNA polymerase-DNA complex have also been obtained. This has been possible by (1) the use of specially modified SFM tips with a consistent radius of curvature of 10 nm or less, to minimize the amount of image distortion introduced by the finite dimensions of commercially available tips, (2) the optimization of a method to deposit and bind DNA molecules to the mica surface in a stable fashion, and (3) careful control of the sample humidity, to prevent solvation of the molecules and detachment from the surface by the scanning tip or stylus. Contact forces in the range of a few nanonewtons are routinely possible in air and in the presence of residual humidity. The spatial resolution of the images appears determined by the radius of curvature of the modified styli, which can be estimated directly from the apparent widths of the DNA molecules in the images.     

Atomic force microscopy of macromolecular interactions.

The introduction of functional imaging tools and techniques that operate at molecular-length scales has provided investigators with unique approaches to characterizing biomolecular structure and function relationships. Recent advances in the field of scanning probe techniques and, in particular, atomic force microscopy have yielded tantalizing insights into the dynamics of protein self-assembly and the mechanics of protein unfolding.     

Atomic force microscopy imaging of DNA under macromolecular crowding conditions

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl 2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt.     

Atomic force microscopy captures MutS tetramers initiating DNA mismatch repair

In spite of extensive research, the mechanism by which MutS initiates DNA mismatch repair (MMR) remains controversial. We use atomic force microscopy (AFM) to capture how MutS orchestrates the first step of E. coli MMR. AFM images captured two types of MutS/DNA complexes: single-site binding and loop binding. In most of the DNA loops imaged, two closely associated MutS dimers formed a tetrameric complex in which one of the MutS dimers was located at or near the mismatch. Surprisingly, in the presence of ATP, one MutS dimer remained at or near the mismatch site and the other, while maintaining contact with the first dimer, relocated on the DNA by reeling in DNA, thereby producing expanding DNA loops. Our results indicate that MutS tetramers composed of two non-equivalent MutS dimers drive E. coli MMR, and these new observations now reconcile the apparent contradictions of previous 'sliding' and 'bending/looping' models of interaction between mismatch and strand signal.     

Atomic force microscopy and chemical force microscopy of microbial cells

Over the past years, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of microbial cells with nanometer resolution, and under physiological conditions. Moreover, chemical force microscopy (CFM) and single-molecule force spectroscopy have enabled researchers to map chemical groups and receptors on cell surfaces, providing valuable insight into their structure-function relationships. Here, we present protocols for analyzing spores of the pathogen Aspergillus fumigatus using real-time AFM imaging and CFM. We emphasize the use of porous polymer membranes for immobilizing single live cells, and the modification of gold-coated tips with alkanethiols for CFM measurements. We also discuss recording conditions and data interpretation, and provide recommendations for reliable experiments. For well-trained AFM users, the entire protocol can be completed in 2-3 d.     

Atomic force microscopy of hydrated phosphatidylethanolamine bilayers.

We present images of the polar or headgroup regions of bilayers of dimyristoyl-phosphatidylethanolamine (DMPE), deposited by Langmuir-Blodgett deposition onto mica substrates at high surface pressures and imaged under water at room temperature with the optical lever atomic force microscope. The lattice structure of DMPE is visualized with sufficient resolution that the location of individual headgroups can be determined. The forces are sufficiently small that the same area can be repeatedly imaged with a minimum of damage. The DMPE molecules in the bilayer appear to have relatively good long-range orientational order, but rather short-range and poor positional order. These results are in good agreement with x-ray measurements of unsupported lipid monolayers on the water surface, and with electron diffraction of adsorbed monolayers.     

Atomic force microscopy of the myosin molecule.

Atomic force microscopy (AFM) has been used to study the structure of rabbit skeletal muscle myosin deposited onto a mica substrate from glycerol solution. Images of the myosin molecule have been obtained using contact mode AFM with the sample immersed in propanol. The molecules have two heads at one end of a long tail and have an appearance similar to those prepared by glycerol deposition techniques for electron microscopy, except that the separation of the two heads is not so well defined. The average length of the tail (155 +/- 5 nm) agrees well with previous studies. Bends in the myosin tail have been observed at locations similar to those observed in the electron microscope. By raising the applied force, it has been possible locally to separate the two strands of the alpha-helical coiled-coil tail. We conclude that the glycerol-mica technique is a useful tool for the preparation of fibrous proteins for examination by scanning probe microscopy.     

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.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

Atomic force microscopy and proteins

This review briefly introduces the principles of atomic force microscopy (AFM) applied to protein samples. AFM provides three-dimensional surface images of the proteins with high resolution. The advantage of AFM for protein studies is that AFM can visualize directly the molecule under physiological conditions without previous treatment. AFM operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand receptor interactions with the goal of measuring forces at the individual molecule level.     

Atomic force microscopy of insulin single crystals: direct visualization of molecules and crystal growth.

Atomic force microscopy performed on single crystals of three different polymorphs of bovine insulin revealed molecularly smooth (001) layers separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prevented molecular-scale resolution, tapping mode imaging in solution provided molecular-scale contrast that enabled determination of lattice parameters and polymorph identification while simultaneously enabling real-time examination of growth modes and assessment of crystal quality. Crystallization proceeds layer by layer, a process in which the protein molecules assemble homoepitaxially with nearly perfect orientational and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation into growing terraces, and their role in defect formation. These observations demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional imaging modes.     

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.