Atomic Force Microscopy of Long and Short Double-Stranded, Single-Stranded and Triple-Stranded Nucleic Acids

Abstract

Atomic force microscopy (AFM, also called scanning force microscopy) is proving to be a useful technique for imaging DNA. Thus it is important to push the limits of AFM imaging in order to explore both what types of DNA can be reliably imaged and identified and also what substrates and methods of sample preparation are suitable. The following advances in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because they are all capable of intramolecular base pairing and because the DNAs were in a Mg(ll) buffer, which facilitates intramolecular cross-bridging. (ii) AFM images in air of short double-stranded DNA molecules, 100-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of poly (A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persistence length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from phX-174 under the same conditions was 80 nm. (iv) Structures believed to be triple- stranded DNA were seen in samples of poly(dA.poly(dT) and poly (dG).poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Entire molecules of lambda DNA, approx. 16 micron long, were imaged clearly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized silicon, although less clearly than on mica.

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

“…This value is smaller than the canonical value of 0.34 nm͞bp that is assigned to B form DNA; however, it is in a good agreement with the values obtained for duplex RNA measured by AFM (27) and A form RNA (28,29). A deviation in the measured contour length of a worm-like nucleic acid polymer from B form DNA by AFM measurement is often observed (30,31). This observation can be explained by the limitation of AFM in resolving DNA bends in the range of Ϯ4 nm and by the polynomial fitting used to measure the contour length, which induces a systematic error in rounding up sharp bends of the DNA trace.…”

“…Analysis of height distribution of dsRNA and ssRNA was consistent with 0.9 nm Ϯ 0.1 and 0.6 nm Ϯ 0.1 (n ϭ 200), respectively. Although the height of the molecule is influenced directly by the surface treatment of the mica (33), these values are in good agreement with the reported height of dsRNA and ssRNA measured by AFM (31). After scoring all of the Y-shaped DbpA-RNA complexes, we measured the total contour length of the molecules and compared it to the expected calculated mean contour length value that was obtained from Eq.…”

“…This observation can be explained by the limitation of AFM in resolving DNA bends in the range of Ϯ4 nm and by the polynomial fitting used to measure the contour length, which induces a systematic error in rounding up sharp bends of the DNA trace. This effect seems to be enhanced in RNA because of the greater flexibility of the molecule (27,31). Moreover, x-ray fiber data and crystallography of dsRNA indicate that dsRNA adopts the A conformation in solution (28,29).…”

“…L ss was calculated by using two different rises per base values (0.3 and 0.6 nm͞base) obtained from crystallography (29) and AFM (31). The mean contour length distributions of the measured and calculated values are 164 Ϯ 3 nm, 143 Ϯ 4 nm (for 530 nt ϫ 0.3 nm͞base), and 230 Ϯ 4 nm (for 530 nt ϫ 0.6 nm͞base), respectively ( Fig.…”

Fluorescence Near-Field Microscopy of DNA at Sub-10 nm Resolution

“…There are no previous AFM experiments that resolved DNA molecules as short as 15 nm; instead, round images for short DNA molecules were observed in this [ Fig. 4(d)] and previous experiments [28].…”