Bezanilla, M., B. Drake, E. Nudler, M. Kashlev, P. K. Hansma, and H. G. Hansma. 1994. Motion and enzymatic degradation of DNA in the atomic force microscope. Biophys. J. 67:2454-2459
PROBING BIOMOLECULES WITH THE ATOMIC FORCE
MICROSCOPE. Helen G. Hansma, Department of Physics,
University of California, Santa Barbara, CA 931106
The AFM can probe the differences in shape between many types
of biological molecules, such as single-stranded, double-stranded
and triple-stranded DNA, and protein channels in membranes. The
AFM can also track some biological processes, such as enzymes
breaking down DNA, or fibrin clots growing. The AFM tracks these
processes even though the molecules are in aqueous solutions
and are much smaller than the wavelength of light. Since the
AFM is only 11 years old, we are just beginning to discover the
ways it can be used to investigate biological molecules and biological
processes.
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Bezanilla, M., B. Drake, E. Nudler, M. Kashlev, P. K. Hansma, and H. G. Hansma. 1994. Motion and enzymatic degradation of DNA in the atomic force microscope. Biophys. J. 67:2454-2459 | DNA molecules being degraded by the enzyme DNaseI. The same DNA molecules are shown here as they are being cut by the enzyme. The red image shows the DNA slightly cut, and the DNA becomes progressively more cut in the orange through purple images. This enzyme cuts DNA even when the DNA is attached to a flat surface as in this experiment. The enzyme molecules are not visible. |
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| DNA being transcribed by the enzyme RNA polymerase. The enzyme (white spot) binds to the DNA (thin line) After the NTP molecules arrive in the third picture on the top row, the enzyme starts to move along the DNA . As the enzyme moves along the DNA, it uses the NTPs to make RNA (not visible) until it comes to the end of the DNA and falls off in the bottom row of pictures. The DNA continually wiggles around, as you can see from the pictures. The scale bar is in nm (nanometers), which are millionths of a millimeter.
Kasas, S., N. H. Thomson, B. L. Smith, H. G. Hansma, X. Zhu, M. Guthold, C. Bustamante, E. T. Kool, M. Kashlev, and P. K. Hansma. 1997. E. coli RNA polymerase activity observed using atomic force microscopy. Biochemistry. 36:461-468. |
| Drug-induced bending of DNA. The yellow colored DNA molecules in the outer ring don't have drug bound to them, while the molecules in the inner rings all have drug bound. Some DNA molecules are highly bent by the drug, while others are almost as straight as drug-free molecules. The AFM is useful for observing shapes of DNA molecules, even molecules as small as these, which have only 400 base pairs of DNA double helix. Two million of these molecules, straightened out and laid end-to-end, would be one inch long. |
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Hansma, H. G., K. A. Browne, M. Bezanilla, and T.
C. Bruice. 1994. Bending and straightening of DNA induced by the
same ligand: characterization with the atomic force microscope.
Biochemistry. 33:8436-8441
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| DNA double helix and triple helix. The AFM can feel the difference between double helical DNA (blue) and triple helical DNA, which is higher (white).
Hansma, H. G., I. Revenko, K. Kim, and D. E. Laney. 1996. Atomic force microscopy of long and short double-stranded, single-stranded and triple-stranded nucleic acids. Nucleic Acids Res. 24:713-720. |
| The AFM can measure the elasticity of materials. These synaptic vesicles are high (white) in the center in the height image but dark in the center in the hardness image, because their centers are harder than their edges. The vesicles are on a hard surface and are from the electric organ of Torpedo, a marine ray. They are about one ten-thousandth of a millimeter ( 1 micron) in diameter. |
Height Image |
Hardness Image |
Laney, D. E., R. A. Garcia, S. M. Parsons, and H. G. Hansma. 1997. Changes in the elastic properties of cholinergic synaptic vesicles as measured by atomic force microscopy. Biophys. J. 72:806-813.
How does the AFM work? The atomic
force microscope feels the surface of a sample with such a discriminating
touch that it can sometimes even sense the individual atoms on
the surface of a crystal such as gold. The AFM does this by raster-scanning
a small tip back and forth over the sample surface. The tip is
on the end of a cantilever, which deflects when the tip encounters
features on the sample surface. This deflection is sensed with
an optical lever (red line): a laser beam reflecting off the end
of the cantilever onto a segmented photodiode magnifies small
cantilever deflections into large changes in the relative intensity
of the laser light on the two segments of the photodiode. In this
way, the AFM makes a topographic map of the sample surface.
For recent reviews of biological atomic force microscopy, see
This work was supported by NSF MCB 9317466 and Digital Instruments.
