Immobilization of DNA for scanning probe microscopy.

Abstract

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-...

Masking generates contiguous segments of metal-coated and bare DNA for scanning tunneling microscope imaging.

“…The contrast reversibly changed depending on the current setting, although some deterioration of the surface was observed presumably due to interaction with the tip. Negative contrast images have also been reported for plasmid DNA a modified gold surface (14) and for purple membranes deposited on Pt/C-coated mica (19).…”

“…Scanning tunneling micrographs have been reported for bare DNA on substrates in vacuum (1,2), in air (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and in solution (15). DNA bound to gold surfaces, either electrochemically (15) or by chemical modification of the substrate (14), can be imaged reproducibly, but the high-resolution views of DNA dried on untreated surfaces are difficult to repeat.…”

“…DNA bound to gold surfaces, either electrochemically (15) or by chemical modification of the substrate (14), can be imaged reproducibly, but the high-resolution views of DNA dried on untreated surfaces are difficult to repeat. In addition, artifacts have been reported on highly oriented pyrolytic graphite (HOPG), a commonly used substrate (16,17).…”

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

“…It should be taken into consideration that the magnitude of the bending angle may be underestimated due to the possible stretching of the DNA molecules induced by forces involved during the drying process after deposition (22). However, control experiments using mica chemically modified with Mg2+, which has been shown to increase the binding force between mica and DNA and thereby should decrease the DNA stretching during drying (23,24), showed a comparable distribution of bending angles after binding of Jun (data not shown). Furthermore, if the observed bend-angle distribution is to be accounted for by random thermal fluctuations, these should be isotropic and thus centered at a mean value of 0 = 00.…”

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

“…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).…”