Most methods for the study of DNA structure have both advantages and disadvantages. Crystallography can give high resolution, but a crystal is required, and packing forces and dehydration present potential sources of artifacts. NMR is carried out in solution, but structures are frequently underdetermined. The lack of long distances is a problem, and this is particularly acute for a linear polymer such as DNA. Both crystallography and NMR are long term projects, requiring months or years of effort. Electron microscopy is satisfying to the less physically inclined investigator, as it feels rather more direct and immediate, yet as a source of structural data it is fraught with potential difficulties. Chief amongst these are dehydration artifacts, radiation damage and forces involved in absorption of samples to the grid. The development of scanning tunneling microscopy (sTM)(~)offers a novel type of microscopy of high resolution, that can be performed without dehydration or sample degradation. The technique of STM is very new; it was the subject of the Nobel rize for physics in 1986. The principle is very simple(2. In the STM an ultrafine conducting probe tip (ideally terminating in a single atom) is placed extremely close to the sample to be studied. If a small bias voltage is applied between a specimen and tip, then a tiny current will flow due to tunneling, the magnitude of which depends the sample-tip separation. The movement of the tip is controlled with a precision in the Angstrom range, by means of a piezoelectric device. This is achieved by an electrical voltage that is connected by a feedback circuit to the tunneling current. As the tip is scanned across the sample, the tunneling current is kept constant by adjusting the voltage to the piezoelectric element. The fluctuation of this voltage therefore represents a record of the surface profile of the sample as the tip moves up and down as it tracks along, maintaining a constant tunneling current. By performing a succession of scans, a surface can be mapped out. In principle, the STM can give very high resolution, since the tunneling current is a sensitive function of the distance between tip and sample, and remarkable images of the surfaces of materials such as metals, semiconductors and graphite have been obtained.
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Potentially the method offers a number of advantages for biological specimens, for samples can be studied under water, and radiation damage is not a problem. However, a number of other problems have limited the success with which the STM has been applied to samples of biological interest. Ideally, for the tunneling to occur, the sample should conduct electrons, but biomolecules do not do this efficiently. One way to achieve this is to apply a thin coating of a metal alloy, as was done in a study of a RecA-DNA filament(3), but this clearly reduces the ultimate resolution. Another approach is to use a metal replica of the specimen. However, if the specimen is placed on a conducting surface, such as gold or graphite, it seems that the biomolecule exhibits sufficient conduction for tunneling to occur, although the mechanism is still unclear. RecADNA filaments have been studied on a platinum-carbon film using STM, and images closely comparable with those of the coated samples obtained(4). There have been a number of recent studies of DNA and other nucleic acids using the STM. Studies of airdried DNA and RNA samples that had undergone lateral association gave measurements of periodicities that were interpreted in terms of helical parameterd5), and comparison with crystallographic values suggested that some shrinkage had occurred during sample preparation. This is most probably due to dehydration. Lindsay and his colleagues have studied duplex DNA under water@),and present images having the appearance of grooved helices. Some of the most remarkable images have been presentedc7) for poly(dG-meSdC). poly(dG-me5dC) in the left-handed Z conformation, air dried on a support of highly oriented pyrolytic graphite. The results seem to show very clearly a left-handed double helix, although once again some shrinkage seems to have resulted from the dehydration. A feature of the images presented is the presence of two deep grooves, yet in Z-DNA the major groove is not a groove at all, but is convex due to displacement of the helix axis into the minor groove. In fact, the STM image of Z DNA is a much better fit to its electrostatic surface, raising the question of what the technique really ‘sees’. Since electron conduction is required, perhaps it is not so surprising that the results are related to the local electronic properties of the molecule studied, and indeed STM has been applied to the study of orbital structure on surfaces in non-biological samples. If a straightforward probing of structure is required, then the related technique of atomic force microscopy, that does not involve electron conduction, might be employed. One of the most recent applications of STM to DNA concerned single-stranded polydA on pyrolytic graphite. Regularly repeating images were obtained, inte reted in terms of an ordered, extended structure( ). There was a repeated structure with a spacing of 6 A that was proposed to be the purine ring, and in some images this was divisible into two domains, postulated to be the five and six membered rings
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comprising the purine nucleus. Dunlap and Bustamante suggest that images of purines and pyrimidines could be differentiated, and speculate that ultimately it might be possible to sequence DNA by this technique. The idea of sequencing DNA by microscopy has a long history, and has not yet been successful. Moreover, any such technique will compete with a number of highly efficient methods. However, I imagine that the availability of considerable funding for sequencing methods from the genome project must focus minds in this direction. STM is a new technique, still in its first decade. So far its application to nucleic acids has not revealed any startling new insights, but we must give it time. I am mindful of the development of NMR imaging, that seemed to progress from hazy images of concentric water cylinders to remarkable cross-sections of brains in an astonishingly short period. There remain fundamental problems associated with the application of STM to biology, but I am sure progress will be made.
References 1 BINNIG, G., ROHRER, H., GERBER, C. AND WIEBEL, E. (1982). Surface studies by scanning tunneling microscopy. Phys Rev Lett 49, 57-60. 2 HANSMA, P. K . , ELINGS,V. B . , MARTI,0. AND BRACKER, C. E. (1988). Scanning tunneling microscopy and atomic force microscopy: applications to biology and technology. Science 242, 209-216. 3 AMREIN, M., STASIAK, A , , GROSS, H., STOLL, E. AND TRAVAGLINI. G . (1988). Scanning tunneling microscopy of recA-DNA complexes coated with a conducting film. Science 240, 514-516. 4 AMREIN. M., DURR,R., STASIAK, A,, GROSS, H. AND TRAVAGLINI, G . (1989). Scanning tunneling microscopy of uncoated recA-DNA complexes. Science 243, 1708-1711. 5 LEE, G., A R S C O P. ~ , G., BLOOMFIELD, V. A. AND EVANS,D. F. (1989). Scanning tunneling microscopy of nucleic acids. Science 244, 475-477. 6 LINDSAY, S. M., THUNDAT, T., NAGAHARA, L . , KNIPPING, U . AND RILL,R. L. (1989). Images of the DNA double helix in water. Science 244, 1063-1064. 7 A R S C OP. ~ , G., LEE, G.. BLOOMFIELD, V. A . AND EVANS,D . F. (1989). Scanning tunnelling microscopy of Z-DNA. Nature 339,484-486. 8 D U N I A PD. . D. AND BUSTAMANTE, c. (1989). Images of single-stranded nucleic acids by scanning tunnelling microscopy. Nature 342, 204-206.
David M. J. Lilley is at the Department of Biochemistry, The University, Dundee DD1 4HN, UK.
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Potentially the method offers a number of advantages for biological specimens, for samples can be studied under water, and radiation damage is not a problem. However, a number of other problems have limited the success with which the STM has been applied to samples of biological interest. Ideally, for the tunneling to occur, the sample should conduct electrons, but biomolecules do not do this efficiently. One way to achieve this is to apply a thin coating of a metal alloy, as was done in a study of a RecA-DNA filament(3), but this clearly reduces the ultimate resolution. Another approach is to use a metal replica of the specimen. However, if the specimen is placed on a conducting surface, such as gold or graphite, it seems that the biomolecule exhibits sufficient conduction for tunneling to occur, although the mechanism is still unclear. RecADNA filaments have been studied on a platinum-carbon film using STM, and images closely comparable with those of the coated samples obtained(4). There have been a number of recent studies of DNA and other nucleic acids using the STM. Studies of airdried DNA and RNA samples that had undergone lateral association gave measurements of periodicities that were interpreted in terms of helical parameterd5), and comparison with crystallographic values suggested that some shrinkage had occurred during sample preparation. This is most probably due to dehydration. Lindsay and his colleagues have studied duplex DNA under water@),and present images having the appearance of grooved helices. Some of the most remarkable images have been presentedc7) for poly(dG-meSdC). poly(dG-me5dC) in the left-handed Z conformation, air dried on a support of highly oriented pyrolytic graphite. The results seem to show very clearly a left-handed double helix, although once again some shrinkage seems to have resulted from the dehydration. A feature of the images presented is the presence of two deep grooves, yet in Z-DNA the major groove is not a groove at all, but is convex due to displacement of the helix axis into the minor groove. In fact, the STM image of Z DNA is a much better fit to its electrostatic surface, raising the question of what the technique really ‘sees’. Since electron conduction is required, perhaps it is not so surprising that the results are related to the local electronic properties of the molecule studied, and indeed STM has been applied to the study of orbital structure on surfaces in non-biological samples. If a straightforward probing of structure is required, then the related technique of atomic force microscopy, that does not involve electron conduction, might be employed. One of the most recent applications of STM to DNA concerned single-stranded polydA on pyrolytic graphite. Regularly repeating images were obtained, inte reted in terms of an ordered, extended structure( ). There was a repeated structure with a spacing of 6 A that was proposed to be the purine ring, and in some images this was divisible into two domains, postulated to be the five and six membered rings
T
comprising the purine nucleus. Dunlap and Bustamante suggest that images of purines and pyrimidines could be differentiated, and speculate that ultimately it might be possible to sequence DNA by this technique. The idea of sequencing DNA by microscopy has a long history, and has not yet been successful. Moreover, any such technique will compete with a number of highly efficient methods. However, I imagine that the availability of considerable funding for sequencing methods from the genome project must focus minds in this direction. STM is a new technique, still in its first decade. So far its application to nucleic acids has not revealed any startling new insights, but we must give it time. I am mindful of the development of NMR imaging, that seemed to progress from hazy images of concentric water cylinders to remarkable cross-sections of brains in an astonishingly short period. There remain fundamental problems associated with the application of STM to biology, but I am sure progress will be made.
References 1 BINNIG, G., ROHRER, H., GERBER, C. AND WIEBEL, E. (1982). Surface studies by scanning tunneling microscopy. Phys Rev Lett 49, 57-60. 2 HANSMA, P. K . , ELINGS,V. B . , MARTI,0. AND BRACKER, C. E. (1988). Scanning tunneling microscopy and atomic force microscopy: applications to biology and technology. Science 242, 209-216. 3 AMREIN, M., STASIAK, A , , GROSS, H., STOLL, E. AND TRAVAGLINI. G . (1988). Scanning tunneling microscopy of recA-DNA complexes coated with a conducting film. Science 240, 514-516. 4 AMREIN. M., DURR,R., STASIAK, A,, GROSS, H. AND TRAVAGLINI, G . (1989). Scanning tunneling microscopy of uncoated recA-DNA complexes. Science 243, 1708-1711. 5 LEE, G., A R S C O P. ~ , G., BLOOMFIELD, V. A. AND EVANS,D. F. (1989). Scanning tunneling microscopy of nucleic acids. Science 244, 475-477. 6 LINDSAY, S. M., THUNDAT, T., NAGAHARA, L . , KNIPPING, U . AND RILL,R. L. (1989). Images of the DNA double helix in water. Science 244, 1063-1064. 7 A R S C OP. ~ , G., LEE, G.. BLOOMFIELD, V. A . AND EVANS,D . F. (1989). Scanning tunnelling microscopy of Z-DNA. Nature 339,484-486. 8 D U N I A PD. . D. AND BUSTAMANTE, c. (1989). Images of single-stranded nucleic acids by scanning tunnelling microscopy. Nature 342, 204-206.
David M. J. Lilley is at the Department of Biochemistry, The University, Dundee DD1 4HN, UK.
