The investigators recently perfected methods for imaging DNA molecules with an STM while the molecules are covered by aqueous electrolytes. The molecules are deposited onto a single-crystal gold surface using electrochemical methods. The gold has been thoroughly characterized and (prior to deposition of DNA molecules) shows the same fine (0.1 angstrom) structure as observed in ultrahigh vacuum. Imaging is carried out in situ and under electrochemical potential control. High resolution images are obtained. They are free from the artifacts that have plagued similar attempts to image DNA on graphite substrates. Double helical and single-stranded DNA can be imaged quite easily. Stacked aggregates of the bases can be imaged at near-atomic resolution. In particular, the purines, which self-associate into DNA-like chains under water, yield images in which each base is seen clearly.The contrast of the bases in these images depends on their electrochemical properties. Thymine produces distinctive images when the substrate potential is raised so as to drive a reversible electron transfer reaction. In this proposal, the investigators outline a program for developing this technology as an alternative method for sequencing DNA.If the bases could indeed be routinely identified by their contrast in STM images made in controlled electrochemical conditions, then DNA polymers could be imaged as rapidly as the molecule could be scanned (potentially hundreds of bases per second). Furthermore, although many technical obstacles need to be overcome to demonstrate a practical method for rapid sequencing, there is no intrinsic limit to the length of the polymer that could be sequenced by these methods. In the initial feasibility study, the investigators' specific aims are as follows: (1) The investigators will examine STM images of nucleosides and nucleotides in controlled electrochemical conditions with the goal of establishing the factors involved in the electrochemical adsorption of DNA onto gold, and establishing the conditions that give optimum electronic contrast for distinguishing the bases. (2) The investigators will examine single-stranded DNA oligomers with the goal of optimizing resolution so that single bases are resolved routinely. They will also study imaging in denaturing conditions and in the presence of heavy metal labels. (3) They will continue to develop atomic force microscopy (AFM) for DNA imaging, both to check their STM methods and to develop better methods for imaging DNA- protein complexes.
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| Lyubchenko, Y L; Blankenship, R E; Gall, A A et al. (1996) Atomic force microscopy of DNA, nucleoproteins and cellular complexes: the use of functionalized substrates. Scanning Microsc Suppl 10:97-107;discussion 107-9 |
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