Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.     

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.     

Stretching single molecules into novel conformations using the atomic force microscope

A dense network of interconnected proteins and carbohydrates forms the complex mechanical scaffold of living tissues. The recently developed technique of single molecule force spectroscopy using the atomic force microscope (AFM) has enabled a detailed analysis of the force-induced conformations of these molecules and the determinants of their mechanical stability. These studies provide some of the basic knowledge required to understand the mechanical interactions that define all biological organisms.     

Affinity imaging of red blood cells using an atomic force microscope.

We used an atomic force microscope (AFM) to produce an image of a mixed layer of group A and O red blood cells with a contrast based only on the measured strength of a specific receptor-ligand pair. The image was obtained by measuring and plotting for each image pixel the adhesion force between the mixed RBC layer and the AFM tip functionalized with Helix pomatia lectin. The high specificity of that lectin for the N -acetylgalactosamine-terminated glycolipids present in the membrane of group A RBCs enabled us to discriminate between the two cell populations and to produce an image based on affinity contrast. The rupture force of the adhesion events leading to the image formation were quantitatively analyzed and compared to rupture forces measured with the same AFM tip on N-acetylgalactosamine tethered to agarose beads. The mean rupture force was found to be 65 pN when measured on the group A RBCs and 35 pN on the agarose beads. These results show that the adhesion, mediated by only a few receptor-ligand pairs, produces sufficient contrast for the affinity image formation.     

Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope.

To achieve high-resolution topographs of native biological macromolecules in aqueous solution with the atomic force microscope (AFM) interactions between AFM tip and sample need to be considered. Short-range forces produce the submolecular information of high-resolution topographs. In contrast, no significant high-resolution information is provided by the long-range electrostatic double-layer force. However, this force can be adjusted by pH and electrolytes to distribute the force applied to the AFM tip over a large sample area. As demonstrated on fragile biological samples, adjustment of the electrolyte solution results in a local reduction of both vertical and lateral forces between the AFM tip and proteinous substructures. Under such electrostatically balanced conditions, the deformation of the native protein is minimized and the sample surface can be reproducibly contoured at a lateral resolution of 0.6 nm.     

Biomolecular force measurements and the atomic force microscope

The atomic force microscope (AFM) is a surface-sensitive instrument capable of imaging biological samples at nanometer resolution in all environments including liquids. The sensitivity of the AFM cantilever, to forces in the pico Newton range, has been exploited to measure breakaway forces between biomolecules and to measure folding-unfolding forces within single proteins. By attaching specific antibodies to cantilevers the simultaneous imaging of target antigens and identification of antigen-antibody interactions have been demonstrated.     

Method for orienting DNA molecules on mica surfaces in one direction for atomic force microscopy imaging.

An efficient method was developed to stretch DNA molecules on an atomically flat surface for AFM imaging. This method involves anchoring DNA molecules from their 5' ends to amino silanized mica surfaces. N-Succinimidyl6-[3'-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP), a heterobifunctional cross-linker with a flexible spacer arm was used for this purpose. Immobilization was carried out by introducing a thiol group to the 5' end of DNA by PCR. Thiolated molecules were then reacted with the cross linker to conjugate with its 2-pyridyl disulphide group via sulfhydryl exchange. The resulting complex was deposited on amino silanized mica where NHS-ester moiety of the cross linker reacted with the primary amino group on the surface. Samples were washed by a current of water and dried by an air jet in one direction parallel to the surface. DNA molecules were fully stretched in one direction on imaging them by AFM.     

Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope.

The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions. Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays. In this study, we have imaged in real-time individual collagen I molecules and their proteolysis by Clostridium histolyticum collagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM). We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules. The proteolysis of collagen molecules was inhibited by reduced calcium and acidification. Results from AFM study of collagen proteolysis are consistent with SDS-PAGE biochemical assays. The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays. This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale. A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level. Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.     

Visualization of hemiknot DNA structure with an atomic force microscope

The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)_n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane–mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.     

Hydration force in the atomic force microscope: A computational study.

Using a hard sphere model and numerical calculations, the effect of the hydration force between a conical tip and a flat surface in the atomic force microscope (AFM) is examined. The numerical results show that the hydration force remains oscillatory, even down to a tip apex of a single water molecule, but its lateral extent is limited to a size of a few water molecules. In general, the contribution of the hydration force is relatively small, but, given the small imaging force ( approximately 0.1 nN) typically used for biological specimens, a layer of water molecules is likely to remain "bound" to the specimen surface. This water layer, between the tip and specimen, could act as a "lubricant" to reduce lateral force, and thus could be one of the reasons for the remarkably high resolution achieved with contact-mode AFM. To disrupt this layer, and to have a true tip-sample contact, a probe force of several nanonewtons would be required. The numerical results also show that the ultimate apex of the tip will determine the magnitude of the hydration force, but that the averaged hydration pressure is independent of the radius of curvature. This latter conclusion suggests that there should be no penalty for the use of sharper tips if hydration force is the dominant interaction between the tip and the specimen, which might be realizable under certain conditions. Furthermore, the calculated hydration energy near the specimen surface compares well with experimentally determined values with an atomic force microscope, providing further support to the validity of these calculations.     

A method for anchoring round shaped cells for atomic force microscope imaging.

More and more researchers are interested in imaging living (Henderson, 1994) or fixed cells in their natural environment using the atomic force microscope (AFM). However, the AFM tip interacts strongly with the sample, and its z range freedom is limited to a few micrometers. This means that the cells to be imaged have to be strongly attached to the substrate, and imaging is restricted to cells having a flattened shape. Here we propose a simple and inexpensive solution to overcome these limitations. The method we propose is trapping living round shaped cells in a Millipore filter with a pore size comparable to the dimensions of the cell. The highest part of some of the blocked cells protrude through the holes of the filter and can this way be easily observed using the AFM without detachment.     

Amyloid under the atomic force microscope

The atomic force microscope (AFM) is a versatile instrument that can be used to image biological samples at nanometre resolution as well as to measure inter and intra-molecular forces in air and liquid environments. This review summarises the use of AFM applied to protein and peptide self-assembly systems involved in amyloid formation. The technical principles of the AFM are outlined and its advantages and disadvantages are highlighted and discussed in the context of the rapidly developing field of amyloid research.     

Mapping interaction forces with the atomic force microscope.

Force curves were recorded as the sample was raster-scanned under the tip. This opens new opportunities for imaging with the atomic force microscope: several characteristics of the samples can be measured simultaneously, for example, topography, adhesion forces, elasticity, van der Waals, and electrostatic interactions. The new opportunities are illustrated by images of several characteristics of thin metal films, aggregates of lysozyme, and single molecules of DNA.     

Observation of living cells using the atomic force microscope.

We used an atomic force microscope (AFM) to image samples immersed in a fluid in order to study the dynamic behavior of the membranes of living cells. AFM images of cultured cells immersed in a buffer were obtained without any preliminary preparation. We observed surface changes and displacements which suggest that the cells were still alive during the measurements. Some membrane details imaged with the AFM have also been observed using a scanning electron microscope and their dynamic behavior has been confirmed by microcinematography. We believe that the AFM will offer new insights into the exploration of dynamic changes affecting cell membranes.     

Structure of branched DNA molecules: gel retardation and atomic force microscopy studies.

DNA heteroduplexes as models for slipped strand DNA have been analyzed by polyacrylamide gel migration and atomic force microscopy (AFM). All heteroduplexes containing one hairpin or loop have reduced electrophoretic mobilities compared with that expected for their molecular weights. The retarded gel mobility correlates with the formation of a sharp kink detected by AFM. Increasing the hairpin length from 7 bp to 50 bp results in a monotonous decrease in gel mobility of heteroduplexes. This secondary retardation effect appears to depend only on the hairpin size since the AFM data show no dependence of the kink angle on the hairpin length. Heteroduplex isomers with a loop or hairpin in opposite strands migrate with distinct mobilities. Analysis of gel migration of heteroduplexes with altered hairpin orientations as well as of truncated heteroduplexes indicates that the difference in mobility is due to an inherent curvature in one of the long arms. This is confirmed by the end-to-end distance measurements from AFM images. In addition, significant variation of the end-to-end distances is consistent with a dynamic structure of heteroduplexes at the three-way junction. Double heteroduplexes containing one hairpin in each of the complementary strands also separate in a gel as two isomers. Their appearance in AFM showed a complicated pattern of flat representations of the three-dimensional structure and may indicate a certain degree of interaction between complementary parts of the hairpins that are several helical turns apart.     

Imaging cells with the atomic force microscope

Different types of cells have been imaged with the atomic force microscope. The morphology of the archaebacterium Halobacterium halobium in its dry state was revealed. On a leaf of the small Indian tree Lagerstroemia subcostata a stoma was imaged. The lower side of a water lily leaf was imaged in water showing features down to 12 nm. Finally, fixed red and white blood cells were imaged in buffer showing features down to 8 nm. The images demonstrate that atomic force microscopy can provide high-resolution images of cell surfaces under physiological conditions.     

Detecting ultraviolet damage in single DNA molecules by atomic force microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m(2) of UVC and 165 kJ/m(2) of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced approximately 0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m(2) of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.     

Separation of triplet repeat DNA by capillary electrophoresis and the conformational analysis by atomic force microscope.

We investigated the relationship between electrophoretic behaviors and higher order structures of triplet repeat DNA fragments by means of capillary electrophoresis and atomic force microscopy (AFM). It was suggested that the mobility difference between triplet repeat DNA and random sequence DNA should be correlated to differences in their dynamic conformation.