Case study: Synthesis of triple-stranded DNA[1]

Double-stranded poly(dG)–poly(dC) and triple-stranded poly(dG)–poly(dG)–poly(dC) DNA were deposited on the surface of HOPG and visualized using AFM. The high resolution attained by HRC enabled to detect single-stranded regions in double-stranded poly(dG)–poly(dC) and double-stranded and single-stranded regions in poly(dG)–poly(dG)–poly(dC) triplexes, as well as to resolve the helical pitch of the triplex molecules. The reaction of G-strand extension in poly(dG)–poly(dC) by the Klenow exo⁻ fragment of DNA polymerase I was followed. The dynamics of the triplex synthesis on a molecular level was monitored by identifying double- and triple-stranded regions along the synthesized poly(dG)–poly(dG)–poly(dC) molecules.

As seen in figure 1, three different structural motifs, with heights of 0.4 ± 0.1 nm, 1.0 ± 0.1 nm and 2.0 ± 0.1 nm, respectively, can clearly be distinguished along the molecules. It should also be noted that, when imaged with commercial tips, no clear distinction of the structural alterations along the triplex molecules could be obtained. This attests to the higher spatial resolution of HRC.

Figure 1. High-resolution AFM image of poly(dG)–poly(dG)–poly(dC) triplex DNA. Notice the single-strand (dark blue), double-strand (light blue) and triple-strand (red) regions in the molecules. The bottom-right inset is a cross-section of triplex, dsDNA and ssDNA regions in the molecule, taken at the short unmarked lines in the image. Bottom-left diagrams present schematic views of three types of molecules, as indicated by the arrows: 1, full-length or ‘completed’ triplex molecule; 2, ‘partially-synthesized’ triplex, containing both triplex and dsDNA fragments; 3, ‘over-synthesized’ triplex, containing a single-stranded fragment at the end of the molecule.

The dependences of the height and the width of different parts of the DNA molecules on the number of strands of which they are comprised are presented in figure 2. Both the molecular height and the width depend linearly on the number of strands composing the DNA. The measured height on HOPG is higher than the height on mica. The apparent width, measured by HRC on HOPG, is consistently higher than the molecular height, having the same slope. This constant difference (3.3 nm) is at least partially due to the convolution of the real molecule’s width with the tip radius. Therefore it is also plausible that the molecule bound to the surface does not present a round cross-section to the AFM; its deconvoluted width is about 1–1.5 nm greater than its height. Similar AFM images of ssDNA and dsDNA done on HOPG using conventional Si probes have yielded much higher values. It should be noted that the small radius of HRC allows such small values of DNA width to be measured, thus allowing to visualize morphological features of the molecules.

Figure 2. Dependence of the height (bottom traces) and the width (top traces) of molecules on the number of strands composing the DNA. Bottom trace (squares): DNA height on mica. Circles: DNA height on HOPG. Diamonds: DNA width on HOPG. Top trace (triangles): DNA width on HOPG, using conventional AFM tip.

To obtain more insight into the synthesis process, successive stages in the reaction were imaged by sampling the assay after different durations, from 0 (before the addition of the enzyme) to 6 h. At each time the sampled molecules were deposited on HOPG and visualized by AFM using HRC (see figure 3). In this way a series of ‘snapshots’ of successive stages within the synthesis process was obtained. The length of the triplex fragment (seen as a bright fragment) increases with the synthesis time, while the length of the remaining double-stranded fragment (seen as a narrower and darker fragment) decreases. As before, the difference in the brightness in the image of the fragments along the synthesized molecule is due to the difference in the height between the triplex and the dsDNA, 2 ± 0.1 nm and 1.0 ± 0.1 nm, respectively.

Figure 3. High-resolution ‘snapshot’ AFM images of the products of poly(dG–dG)–poly(dC) synthesis. The AFM images were taken after duration of 0 h (a), 0.5 h (b), 1 h (c), 2 h (d), 3 h (e) and 6 h (f) from the beginning of the synthesis. A schematic presentation of the intermediate products of the synthesis is shown at the bottom of each picture (the G-strand de novo is marked in bold).

The detailed molecular morphology of the triplex molecules along their axis was also investigated using the same HRC. Periodic variations in the height along the molecule were observed, especially in AFM phase images depicting the change of phase of the cantilever oscillations relative to its driving force (figure 4). The length of the periodic motif (an example of which is indicated in the figure by arrows) is approximately 3.4 ± 0.9 nm. This corresponds, within experimental error, to the length of periodic motif of the triplex DNA (3.9 ± 0.1 nm) determined using NMR and x-ray diffraction studies as well as in contact-mode AFM studies of highly packed (liquid-crystal-like) dsDNA in a liquid cell (3.4 ± 0.4 nm). The relatively wide distribution of the length of the periodic motif observed is due to the interaction between the molecule and the substrate, as well as to the dry ambient in which the molecule was imaged. This image also illustrates the fine resolution capabilities of HRC.

Figure 4. Left: high-resolution AFM phase image of poly(dG)–poly(dG)–poly(dC) triplex DNA. The top graph shows a cross-section of the image along the white line (at the bottom part of the image). Right: histogram of distances between adjacent peaks on cross-sections taken on many molecules; the average distance is 3.4 nm (SD = 0.9 nm).

One of the most interesting advantages of AFM is its ability to provide high-resolution images of biomolecules under physiological conditions. The triplex DNA under water ambient (Tris buffer, pH = 7.0) in a wet AFM cell was imaged using HRC (figure 5). In spite of the higher noise in this image (including some horizontal fluctuations), the DNA molecule is well imaged and the average measured width of the molecule is 2.5–3 nm, as shown in the typical cross-section. These values represent a marked improvement over standard AFM measurements made under liquid, typically showing a width of 7–10 nm.

Figure 5. AFM images of poly(dG)–poly(dG)–poly(dC) triplex DNA deposited on HOPG and imaged under water with HRC tip in tapping mode. The average width of the DNA molecule is 2.5–3 nm and its height is 2.0 ± 0.2 nm. Left inset: magnified view of a triplex molecule. Right inset: height profile of the molecule, taken along the line shown in the left inset.

The synthesis of the poly(dG)–poly(dC) dsDNA itself is performed in a similar way to the synthesis of the triplex molecule. A high-resolution image of poly(dG)–poly(dC) dsDNA is shown in figure 6. The dsDNA molecules contain fragments (‘loops’) where the two strands are decoupled and can be seen as two single strands not associated with one another. The measured height of the decoupled strands in the loop is approximately equal to 0.4 ± 0.1 nm. Some of the poly(dG)–poly(dC) molecules contain single-strand fragments (‘tails’), the height of which is also approximately equal to 0.4 nm; the height of dsDNA is ~1 nm, as in the previous samples. These images show the advantage of HRC tips, without which such high spatial resolution would not have been possible. Another interesting feature is the inhomogeneous height of double-stranded parts of the molecule. The origin of these is twists and kinks in the molecule, which are visible due to the high spatial resolution.

Figure 6. Left: high-resolution AFM image of poly(dG)–poly(dC) dsDNA. Right: enlarged view of the left part of the molecule. Arrow 1 points to a single-stranded fragment at the end of the molecule and arrow 2 to a region where the two strands are separated, forming a ‘loop’. The structure of each region is shown schematically below the AFM image.

The visualization of DNA by AFM with HRC makes it possible to detect various structural motifs along double-and triple-stranded DNA molecules as well as to resolve the fine structure of the triplex molecules.

The samples were imaged by Nanoscope III (Veeco, USA) and Ntegra (NT-MDT, Russia) AFMs.

Images were kindly given by Dr. D. V. Klinov, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences.

[1] D. Klinov, B. Dwir, E. Kapon, N. Borovok, N. Molotsky, and A. Kotlyar, High-resolution atomic force microscopy of duplex and triplex DNA molecules, Nanotechnology 18 225102 (2007)