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

Abstract

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-...

Structure and dynamics of supercoil-stabilized DNA cruciforms

“…The AP-mica has speci®c surface characteristics that should be considered in understanding the behavior of DNA molecules and the cruciforms in particular at the surface-liquid interface and their interaction with the surface. As discussed in our previous publications (Lyubchenko et al, 1992(Lyubchenko et al, , 1993b(Lyubchenko et al, , 1995Lyubchenko & Shlyakhtenko, 1997), AP-mica is a positively charged surface with uniform and comparatively low surface charge density. There are three main consequences of these characteristics of AP-mica.…”

“…The key in the AFM sample preparation procedure is the use of the functionalized substrate (AP-mica) which permits one to obtain high resolution AFM images of DNA by a one step deposition from a very wide range of ionic conditions (Lyubchenko et al, 1992(Lyubchenko et al, , 1993aLyubchenko & Shlyakhtenko, 1997). This procedure is highly reproducible and allows one to perform imaging in air or in aqueous solutions (Lyubchenko et al, 1992(Lyubchenko et al, , 1993bLyubchenko & Shlyakhtenko, 1997).…”

Conformational transition in DNA on a cold surface

“…The precise nature of the DNA-surface interactions is still unclear so we set out to compare three different methods: (i) AP-mica (14); (ii) nickel-treated mica (15); and (iii) mica treated with Mg alone (18) without the use of HEPES (17). We found that the magnesium treatment causes linear DNA to condense on the surface (an effect not observed with small circles; 18).…”

“…Several different approaches to binding DNA for imaging under buffer have been described in the literature (14)(15)(16)(17)(18). The precise nature of the DNA-surface interactions is still unclear so we set out to compare three different methods: (i) AP-mica (14); (ii) nickel-treated mica (15); and (iii) mica treated with Mg alone (18) without the use of HEPES (17).…”

“…An aliquot of 300 l of DNA solution (2 ng/ l in 0.1 mM Tris-HCl, pH 8) was placed on the mica in the AFM liquid cell and imaged after 10 min. We have found this method to be more reliable than the earlier vapor-based technique (12)(13)(14). For deposition below 25 C, the mica was prepared as described above and, after drying, mounted on the sample plate of the AFM (see below) and then placed in an enclosure submerged in a thermostatted water bath in a cold room (1.8 C).…”