Structure of hydrated oligonucleotides studied by in situ scanning tunneling microscopy.

Abstract

We have used the scanning tunneling microscope (STM) to image several synthetic oligonucleotides adsorbed onto a positively charged Au(111) electrode. The molecules were deposited and imaged in aqueous electrolyte under potential control, a procedure that eliminated the problem of the substrate artifacts that had limited some previous STM studies. Experiments were carried out with two types of single-stranded molecules (11 and 20 bases long) and three types of double-stranded molecules (20 and 61 base pairs and 31 bases with 25 bases paired and 6-base "sticky" ends). The molecules lie along symmetry directions on the reconstructed (23 x \/3-) gold surface, and length measurements indicate that they adopt simple base-stacked structures. The base stacking disances are, within experimental uncertainty, equal to the 0.33 nm measured for polymeric aggregates of stacked purines by direct imaging in identical conditions. The images show features consistent with helical structures. Double helices have a major-groove periodicity that is consistent with a 360 twist.The single helices appear to be more tightly twisted. A simple tunneling model of STM contrast generates good agreement between measured and calculated images.The scanning tunneling microscope (STM) can resolve atoms under water (1) and many images of biopolymers have been reported (2), including images of DNA which appear to show both the major and minor grooves of the double helix (3) and even atomic resolution (4). These images are controversial because they were obtained on graphite substrates, which can mimic DNA (5) even at the atomic scale (6). Dunlap et al. (7) have estimated the conductance of dry DNA in air by studying contiguous segments of metal-coated and bare molecules, concluding that dry DNA is too good an insulator to be imaged by STM. Allison et aL (8) have reported images of plasmid DNA chemically bound to gold and imaged in air. The images are reproducible, but the contrast is variable (it can change from negative to positive). An altogether different approach uses electrochemical methods to bind molecules onto a gold electrode, which is then imaged under a covering layer of electrolyte (9, 10). Reliability has been improved by maintaining potential control of the electrode during imaging (11). The deposition of molecules can be monitored with this process. Thus, in addition to imaging in an aqueous environment, the substrate artifacts that have plagued some other work can be ruled out. Despite these advantages, the full potential of the method was not realized owing to instrumental limitations (12). We have now built a new microscope and have used it to study a number of different oligomers. WeThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.have also verified our interpretation of images in a blinded experiment (A.M.J., T.W.J., J.A.D., A.V., D.R., F.-X.L., and S.M.L., unpubl...

Preparation of DNA and nucleoprotein samples for AFM imaging

“…Note that in Lyubchenko and Shlyakhtenko (1997), high-resolution images were obtained for individual DNA molecules rather than tightly packed DNA molecules (Mou et al, 1995). High-resolution STM images of DNA are primarily obtained for dried samples, although the electrochemical approach was useful for imaging of short DNA molecules (Jing et al, 1993). However, covalent bonding to the surface using thiolated DNA was needed to enhance the resolution of STM images of DNA (Rekesh et al, 1996).…”

Calculation of Hydroxyl Radical Attack on Different Forms of DNA

“…Such simplified model of ssDNA is supported by the fact that homopolynucleotides (e.g. poly(A), poly(C)) can occur as single-stranded helices (1) which were also observed by the scanning tunneling microscope for single stranded oligonucleotides (15). The helical model of ssDNA was also used to calculate cross sections for the electron interaction with single stranded DNA molecule (16).…”

Immobilization and Visualization of DNA and Proteins on Carbon Nanotubes

“…[9] For example, DNA oligonucleotides can be imaged in aqueous solution by adsorption onto a positively charged gold(111) electrode substrate. [8] This has provided images with helical structures and even indications of the major groove periodicity of the double helices. However, reproducible fixation of samples on an atomically even surface is still the primary challenge, and images with atomic resolution are difficult to achieve.…”

“…In these cases, the DNA molecules are immobilized onto even-surfaced substrates such as gold [8] and mica. [9] For example, DNA oligonucleotides can be imaged in aqueous solution by adsorption onto a positively charged gold(111) electrode substrate.…”