Molecular Structure of DNA by Scanning Tunneling Microscopy

Cricenti, A.; Selci, S.; Felici, Anna Candida; Generosi, R.; Gori, E.; Djaczenko, W.; Chiarotti, G.

doi:10.1126/science.2781279

Cited by 79 publications

(26 citation statements)

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

“…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.…”

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confidence: 99%

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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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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Immobilization of DNA for scanning probe microscopy.

Allison

Bottomley

Thundat

et al. 1992

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

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“…The propanol eliminates forces due to the capillary condensation that occurs in ambient conditions, allowing less contact force to be used in imaging. Hansma et al (8) Chemical methods for holding DNA in place have been proposed by us (9) and others (10) and implemented in ambient conditions for STM imaging (11) and AFM imaging (12)(13)(14). Our method for chemically modifying mica substrates uses covalently bound 3-aminopropyltriethoxysilane (APTES) followed by methylation and hydrolysis (12)(13)(14).…”

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confidence: 99%

Atomic force microscopy of long DNA: imaging in air and under water.

Lyubchenko

Shlyakhtenko

Harrington

et al. 1993

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

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169

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We have obtained striking atomic force microscopy images of the intact A bacteriophage genome and of several A restriction fragments both in air and under water. The DNA is unstained and the images are stable under continuous scanning for up to 30 min. Measured contour lengths of fully imaged restriction fragments and intact A DNA are accurate to within a few percent. The key to this development is the use of a process for binding unmodified double-stranded DNA to chemically treated mica surfaces. This procedure leads to strong DNA attachment and yields high-quality images that are stable under repeated scanning, even with the sample submerged in water. This allows normal hydration conditions to be maintained during scanning and in addition leads to a general improvement of image quality. Both the lateral resolution and the contrast increase by a factor of -3 under water.Scanning probe microscopy, which currently includes scanning tunneling microscopy (STM) and atomic force microscopy (AFM), is a structural tool ofgreat potential importance to structural molecular biology. AFM can image thick insulators, whereas STM is limited to small molecules (1, 2). Resolution is limited by the size and geometry ofthe scanning tip (3), by the tendency of the tip to move the DNA (1, 4), and by deformation of the sample (5) and scanning probe (6). Bustamante et al. (7) have overcome the problem of holding DNA in place for AFM scanning with an ionic treatment of the mica surface that allows DNA to be imaged in air under ambient conditions. Hansma et al. (8) Chemical methods for holding DNA in place have been proposed by us (9) and others (10) and implemented in ambient conditions for STM imaging (11) and AFM imaging (12)(13)(14). Our method for chemically modifying mica substrates uses covalently bound 3-aminopropyltriethoxysilane (APTES) followed by methylation and hydrolysis (12)(13)(14). In preliminary scans in air, this method has enabled us to obtain high-quality images of linear and circular DNA (12). In the work reported here, we show that the method can be extended both to unstained DNA molecules as large as the intact A phage genome (48,502 bp) and to scanning DNA under water. DNA images obtained under water not only reflect normal hydration conditions but also show significantly improved lateral resolution over those obtained in air. It therefore appears possible to use AFM for direct conformational studies of nucleic acids and nucleoprotein complexes. MATERIALS AND METHODSThe procedure for mica modification is described in refs. 12-14. Briefly, freshly cleaved strips of mica were left in the APTES atmosphere created by a small pool of APTES in the bottom of a 2-liter glass desiccator left at ambient temperature for 2 h. The methylation procedure was the same as described in ref. 14. The amino groups of APTES are bound covalently to a freshly cleaved mica surface, endowing it with properties similar to an anion exchanger.Modified mica strips were immersed into DNA in Tris'HCl buffer (pH 7) (10 mM Tris.HCl/10-...

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“…The slightly lower values of the dried samples (85 ± 11% of the control) may be due to small changes in structure of the protein or to incomplete solubilization of the AChR from the HOPG surface by the detergent. DISCUSSION STM imaging of biological macromolecules has only recently been applied to nucleic acids (22)(23)(24)(25), globular proteins (26,27), and glycogen (28) …”

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confidence: 99%