Scanning Tunneling Microscopy of Nucleic Acids

G, Lee; Arscott, PG; Bloomfield, Victor A.; Evans, D. F.

doi:10.1126/science.2470146

Cited by 93 publications

(22 citation statements)

References 15 publications

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“…With the exception of a few researchers (3,4), most of the initial results in this field were obtained with highly ordered pyrolytic graphite (HOPG) as the conducting substrate. For example, several researchers have presented images illustrating the capability of the STM to resolve the major and minor grooves of DNA with a wide range of periodicities (5)(6)(7)(8). The possibility of using the STM to distinguish between the purine and pyrimidine bases ofDNA has been illustrated by studies ofpoly(dA) molecules deposited onto HOPG and imaged in air (9).…”

mentioning

confidence: 99%

Graphite: A Mimic for DNA and Other Biomolecules in Scanning Tunneling Microscope Studies

Clemmer

Beebe

1991

Science

296

133

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Highly ordered pyrolytic graphite (HOPG) is the substrate often used in scanning tunneling microscope (STM) studies of biomolecules such as DNA. All of the images presented in this article are of freshly cleaved HOPG surfaces upon which no deposition has occurred. These images illustrate features previously thought to be due to biological molecules, such as periodicity and meandering of "molecules" over steps. These features can no longer be used to distinguish real molecules from features of the native substrate. The feasibility of the continued use of HOPG as a substrate for biological STM studies is discussed.

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mentioning

confidence: 99%

Graphite: A Mimic for DNA and Other Biomolecules in Scanning Tunneling Microscope Studies

Clemmer

Beebe

1991

Science

296

133

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“…Fragments ofnatural and synthetic DNA have been imaged by STM on highly oriented pyrolytic graphite (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and gold (12,13) surfaces. However, because topographic contrast is generally used to locate these extremely fine (2-nm diameters) molecules by scanning probe techniques, the DNA mounting substrate must be extremely flat.…”

mentioning

confidence: 99%

Immobilization of DNA for scanning probe microscopy.

Allison

Bottomley

Thundat

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Reproducible scanning e microscope and atomic force microscope images of entire molecules of uncoated pasmid DNA chemically bound to surfaces are presented. The chemicaily mediated immobilization of DNA to surfaces and subsequent snning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with nning probe microscopes, can be prevented. (12,13) surfaces. However, because topographic contrast is generally used to locate these extremely fine (2-nm diameters) molecules by scanning probe techniques, the DNA mounting substrate must be extremely flat. Surfaces with atomic flatness are ideal but usually lack sites that might anchor DNA either mechanically or chemically, thereby preventing displacement or removal by the scanning probe tip. Attempts to immobilize DNA on surfaces using electrochemical methods (14, 15) and covalent linking (13, 16, 17) of DNA to surfaces have been successful, but to date only images of DNA fragments have been reported. Evidence for improved surface binding came from examination ofa surface frequently imaged with the AFM. Mica, although atomically flat, is an insulator and therefore cannot be imaged with the STM but is well suited for the AFM. By simply drying a drop of a DNA-containing buffer solution onto freshly cleaved mica, one can routinely obtain AFM images of DNA strands within minutes (18-21), perhaps stabilized by hydrogen bonding of Si-OH to DNA. Recently a technique that replaced potassium ions in the mica surface with magnesium ions, promoting stronger binding of DNA to mica resulting in improved AFM images, has been described (22)(23)(24). Since the forces between the probe and the surface are roughly comparable in STM and AFM, the AFM work gave us hope that with a properly modified conducting surface to stabilize DNA, routine imaging of DNA by STM should be possible. Using an approach that allows a thiol group at one end of an organic molecule to bind to a gold surface (25) and a charged head group at the other end to attract and immobilize the polyanionic DNA for STM imaging (26) we present the STM images of entire plasmid molecules. METHODSPlasmid DNA (3204 base pairs, pBS+ from Stratagene) was extracted from Escherichia coli, concentrated by cesium chloride/ethidium bromide equilibrium centrifugation, ethanol precipitated, and resuspended in 10 mM Tris.HCl/0. 1 mM EDTA (TE) at pH 7.5 to a final concentration of 500 gg/ml (27). Circular DNA was relaxed by x-ray (70,000 rads; 1 rad = 0.01 Gy) mediated single-strand nicking of supercoiled molecules. Linearized plasmid was obtained by treatment with the restriction endonuclease Sma I. Agarose gel (0.8-1% agarose) electrophoresis was used to differentiate supercoiled from relaxed plasmid and linearized DNA (27). Relaxed plasmid DNA was radiolabeled by nick-translation in the following reaction mixture: 35 pl of DNA (500 Ag/ml), 5 pl (10x) of NT buffer (500 mM Tris-HCl, pH 7.5/50 mM MgCl2/100 mM 2-mercaptoethanol/1 mg of nuclease-...

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“…In these micrographs, poly r(A-U) contains hairpin loops whose diameter range from 7-10 nm and whose stems are up to 2.5 nm (25 Å) in diameter. Since the diameter of 25 Å is in close agreement with the 24.8 ± 0.8 Å of the A-form double helix of poly (A) • poly (U) (Lee et al 1989, Saenger 1984, it appears that the stems of these hairpins are double stranded. In fact, double helical structures can be seen in several of these stems when they are viewed at the proper angle.…”

Section: Poly R(a-u) Alonementioning

confidence: 55%

Transmission electron microscopy and scanning force microscopy of poly r(A‐U) and poly r(A‐U)‐ethidium bromide

et al. 1997

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Summary:Transmission electron microscopy and scanning force microscopy of negative-stained, carbon-coated replica and mica-adsorbed preparations of 200 µM poly r(A-U) and 50 µM ethidium bromide/200 µM poly r(A-U) have been employed to evaluate ethidium-induced changes in poly r(A-U) topology. Poly r(A-U) alone exhibits elongated conformations 85-115 nm in length that possess a number of hairpin loops as well as single-stranded domains. While the doublestranded domains are found predominately at the base of the hairpin loops (diameter = 5-30 nm), other rod-like (presumably double-stranded) regions ranging from 25-80 nm in length are present in other portions of the poly r(A-U). In contrast with the poly r(A-U) alone, the EB/poly r(A-U) combination appears as a heterogeneous population of condensed structures whose lengths and widths vary from 12-88 nm and 15-45 nm, respectively. These conformational changes are due to a number of factors, including the displacement of ordered water surrounding the poly r(A-U) and charge shielding of the phosphate groups of the poly r(A-U) upon the binding of the ethidium.

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Scanning Tunneling Microscopy of Nucleic Acids

Cited by 93 publications

References 15 publications

Graphite: A Mimic for DNA and Other Biomolecules in Scanning Tunneling Microscope Studies

Graphite: A Mimic for DNA and Other Biomolecules in Scanning Tunneling Microscope Studies

Immobilization of DNA for scanning probe microscopy.

Transmission electron microscopy and scanning force microscopy of poly r(A‐U) and poly r(A‐U)‐ethidium bromide

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