Atomic Force Microscopy Of DNA Research Articles

Atomic Force Microscopy of DNA and DNA-Protein Interactions.

Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometer resolution. For biological applications, one of its key advantages is its ability to visualize the substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and its interactions with proteins.

Stability, resolution, and ultra-low wear amplitude modulation atomic force microscopy of DNA: Small amplitude small set-point imaging

A way to operate fundamental mode amplitude modulation atomic force microscopy is introduced which optimizes stability and resolution for a given tip size and shows negligible tip wear over extended time periods (∼24 h). In small amplitude small set-point (SASS) imaging, the cantilever oscillates with sub-nanometer amplitudes in the proximity of the sample, without the requirement of using large drive forces, as the dynamics smoothly lead the tip to the surface through the water layer. SASS is demonstrated on single molecules of double-stranded DNA in ambient conditions where sharp silicon tips (R ∼ 2–5 nm) can resolve the right-handed double helix.

Atomic force microscopy of DNA at high humidity: irreversible conformational switching of supercoiled molecules

Three topologically different double-stranded DNA molecules of the same size (bps) have been imaged in air on mica using amplitude modulation atomic force microscopy (AM AFM) under controlled humidity conditions. At very high relative humidity (>90% RH), localized conformational changes of the DNA were observed, while at lower RH, the molecules remained immobile. The conformational changes occurred irreversibly and were driven principally by superhelical stress stored in the DNA molecules prior to binding to the mica surface. The binding mechanism of the DNA to the mica (surface equilibration versus kinetic trapping) modulated the extent of the conformational changes. In cases where DNA movement was observed, increased kinking of the DNA was seen at high humidity when more surface water was present. Additionally, DNA condensation behavior was also present in localized regions of the molecules. This study illustrates that changes in the tertiary structure of DNA can be induced during AFM imaging at high humidity on mica. We propose that AM AFM in high humidity will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution.

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.

Atomic force microscopy of DNA self-assembled on a highly oriented pyrolytic graphite electrode surface

Different types of self-assembled DNA networks were obtained and characterized by tapping mode atomic force microscopy (AFM) on highly oriented pyrolytic graphite (HOPG) electrodes. Both native calf thymus DNA and synthetic DNA with specific base sequences were investigated. The freely adsorption of DNA on the surface of HOPG forms two-dimensional networks. The double-stranded DNA molecules may form cross-linking points on double helix chains, forming intermediate attachments. The differences in AFM morphology of these DNA condensations can be related to the intrinsic properties of the DNA, which lead to different interactions with HOPG. Three-dimensional networks were formed on the HOPG under applied electric field. The effects of external electric field on the condensation of calf thymus DNA have been investigated in detail. It led to a proposition that the DNA molecules may be polarized in the electric field, and attached to the HOPG surface via its one end with the phosphate backbone lying or standing on the substrate depending on the applied potential. The AFM morphological changes of DNA in the presence of external electric field provided insight into DNA interactions.

Atomic Force Microscopy of DNA Immobilized onto a Highly Oriented Pyrolytic Graphite Electrode Surface

Magnetic AC mode atomic force microscopy (MAC Mode AFM) was used to characterize the process of adsorption of DNA on a highly oriented pyrolytic graphite (HOPG) electrode surface using different concentrations of DNA and adsorption procedures. AFM of DNA immobilized on the HOPG showed that both single-stranded DNA and double-stranded DNA molecules have the tendency to spontaneously self-assemble from solution onto the solid support and the process was very fast. DNA condensed on the substrate in a tight and well-spread two-dimensional lattice covering the entire surface uniformly. The interaction of DNA with the hydrophobic HOPG surface induced DNA superposition, overlapping, and intra- and intermolecular interactions. The application of a positive potential of 300 mV (vs Ag wire) to the HOPG electrode during adsorption was studied. The applied potential considerably enhanced the robustness and stability to mechanical stress of the DNA films, through multiple electrostatic interactions between the negatively charged hydrophilic sugar-phosphate backbone and the positively charged carbon surface. The characteristics of the DNA films and the apparent height of the network wires were dependent on the DNA concentration and the immobilization procedure. The DNA lattices were held together on the substrate surface only by noncovalent interactions such as hydrogen bonding, base stacking, electrostatic, van der Waals, and hydrophobic interactions.

Surface biology of DNA by atomic force microscopy.

The atomic force microscope operates on surfaces. Since surfaces occupy much of the space in living organisms, surface biology is a valid and valuable form of biology that has been difficult to investigate in the past owing to a lack of good technology. Atomic force microscopy (AFM) of DNA has been used to investigate DNA condensation for gene therapy, DNA mapping and sizing, and a few applications to cancer research and to nanotechnology. Some of the most exciting new applications for atomic force microscopy of DNA involve pulling on single DNA molecules to obtain measurements of single-molecule mechanics and thermodynamics.

Atomic force microscopy of DNA and protein-DNA complexes using functionalized mica substrates.

Atomic force microscopy (AFM; also called scanning force microscopy [SFM]) is a rather novel technique that offers unique advantages in the potential for the very high resolution of DNA and small ligands in the absence of stains, shadows, and labels (,). Furthermore, the scanning can be performed in air or liquid. The latter is particularly important for resolving fully hydrated structures. The AFM is theoretically capable of resolving structural details at the level of atomic dimensions, provided that the specimen is not dynamic.

Recent Highlights from Atomic Force Microscopy of DNA

Seven recent highlights are presented from atomic force microscopy (AFM) of DNA in this lab. The first two involve advances in the observation of enzymatic reactions in near-physiological solutions. E. coli RNA polymerase was observed to process along its DNA template in a series of time-lapse images [S. Kasas, et al., Biochemistry 36, 461 (1997)], and a new small-cantilever atomic force microscope (AFM) imaged DNA degradation by DNase I at rates as fast as two seconds per image. The next five highlights involve structural observations of DNA and DNA-protein complexes, including DNA condensed for gene delivery, sequence-dependent DNA condensation, an AFM assay for RNA polymerase, and AFM evidence for a yeast kinetochore complex that may be involved in holding together sister chromatids during cell division.

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.

Effect of probe force on the resolution of atomic force microscopy of DNA

Experimental results are presented to show that the adhesion force is the single most important limiting factor in high-resolution atomic force microscopy of DNA in air, prepared by the cytochrome-C-assisted spreading method. It is also shown that humidity plays a minor role in the control of probe force. Using a pure carbon film as the substrate to clean the AFM tip prior to imaging, it is demonstrated that 4–6 nm resolution on DNA can be routinely obtained by the atomic force microscope with commercial Si 3N 4 pyramid cantilevers. We also show that in organic solvents a resolution of up to 3 nm can be obtained under optimal conditions.

Recent advances in atomic force microscopy of DNA.

Three advances involving DNA in atomic force microscopy (AFM) are reported here. First a HEPES-Mg buffer has been used that improves the spreading of DNA and provides good DNA coverage with as little as 200-500 picograms per sample. Second, the new "tapping" mode has been used to improve the ease and resolution of AFM-imaging of DNA in air. Finally, AFM images are presented of single-stranded phi X-174 virion DNA with the gene 32 single-stranded binding protein. A summary of the current state of the field and of the methods for preparing and imaging DNA in the AFM is also presented.

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 DNA and bacteriophage in air, water and propanol: the role of adhesion forces.

We have developed a chemical treatment for the mica surface which allows biopolymers to be held in place for atomic force microscopy, even under water, using conventional, untreated force sensing tips. We illustrate the procedure with images of lambda DNA and fd phage. The phage adheres well enough to permit in situ imaging of the adsorption process in water. These experiments yield a mean length for the phage of 883 +/- 72 nm. This compares with a measured length of 883 +/- 33 nm when the phage are imaged after drying following adsorption from water, showing that the effect of dehydration is quite small. Adhesion forces between the force sensing tip and the substrate and the sensing tip and the biomolecules are very different in the three media (air, water and propanol). The apparent height of the phage and the width and height of the DNA depends upon these adhesion forces quite strongly. In contrast, changing the Hookean spring force exerted by the scanning tip makes little difference. These results suggest that the chemical factors involved in adhesion can dominate atomic force images and that the composition of the scanning tip is at least as important a factor as its geometry.