Case study: Single- and double-stranded DNA on mica and HOPG[1]

ssDNA adsorbed on mica modified by Mg²⁺, by 3-aminopropyltriethoxysilane (APTES) or on modified highly oriented pyrolytic graphite was imaged using AFM. ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG ssDNA molecules adopt a conformation without secondary structures. The immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing.

First, Fig. 1 shows typical AFM images of linear dsDNA deposited (a) on mica from a solution containing divalent ions Mg²⁺; (b) on mica treated by APTES; and (c) on modified HOPG. In Fig. 1A are imaged DNA molecules deposited in the presence of Mg²⁺, which show few crossings, because this deposition method permits a 2D equilibration of dsDNA. In Fig. 1B, the DNA molecules have many crossings, because the strong adsorption on APTES-mica is akin to a geometrical projection from 3D conformation to a 2D plane. The 3D properties can be recovered even if adsorbed on a 2D surface. In Fig. 1C images of dsDNA molecules on ‘‘GM’’ modified HOPG are shown: the dsDNA conformation resembles to that of DNA molecules on mica in the presence of Mg²⁺ (Fig. 1A) with few crossings. Thus, dsDNA on ‘‘GM’’ modified HOPG has a certain degree of 2D relaxation.

Fig. 1. AFM images of Lambda dsDNA adsorbed on the surface of: (A) mica in the presence of Mg²⁺; (B) mica modified by APTES; and (C) ‘‘GM’’ modified HOPG. The bars represent 250 nm on all images.

It is not possible to evaluate native ssDNA on the usual deposition substrates for dsDNA, because ssDNA forms many secondary structures. Here, ssDNA deposited on modified mica or on modified HOPG was studied.

Fig. 2A and B display AFM images of ssDNA M13mp18 and heated linear DNA, respectively. In the latter case, the images include ssDNA and rests of non-denaturated dsDNA. In the presence of Mg²⁺, ssDNA and dsDNA molecules are well adsorbed and can be easily identified. However, the ssDNA parts of the molecules are not elongated and have very compact structures with many kinks and nodes due to intra-strand base-pairing, while the dsDNA parts are in almost 2D equilibrium conformation (Fig. 2B). Additionally, Mg²⁺ ions can link two single strands.

Fig. 2C and D represent images of ssDNA M13mp18 and heated linear DNA on APTES modified mica, respectively. The binding of ssDNA on APTES mica is very strong and the conformation of ssDNA is different in comparison to the dsDNA conformation (see Fig. 1B). One can observe that dsDNA part of the molecules has many crossings due to its strong absorption on the APTES surface (Fig. 2D). At the extremities of the double-stranded parts, it is possible to observe the beginning of separation of the double helix (Fig. 2D). The ssDNA parts are compact with many loops and intra-strand base-pairing (Fig. 2C). The height of the paired ssDNA strands is not exactly the same as for dsDNA, probably indicating that these parts do not have the typical double helix structure. ssDNA structure on APTES mica reminds RNA molecules with many loops and base-pairings. In spite of the compact structure of ssDNA, it was possible to estimate its height and width in the loops. For dsDNA the height was 0.7 ± 0.1 nm and the width 12 ± 3 nm; for ssDNA the height was 0.3 ± 0.1 nm and the width 7 ± 2 nm.

Fig. 2. AFM images of: (A) ssDNA M13mp18; (B) heated Lambda dsDNA adsorbed on the surface of mica in the presence of Mg²⁺; (C) ssDNA M13mp18; and (D) heated Lambda dsDNA on mica modified by APTES. The bars represent 250 nm on all images.

In comparison with ssDNA on modified mica, for ssDNA deposited on HOPG very interesting results were obtained. ssDNA molecules do not have secondary structures as in the case of mica deposition. It was possible to observe the individual ssDNA molecules without intra-strand base-pairing (Fig. 3) and the ssDNA molecules are not as extended as in the case of poly-lysine (Fig. 3A and B). It was possible to estimate the height and width of ssDNA molecules. For dsDNA the height was 0.9 ± 0.1 nm and width 12 ± 3 nm; for ssDNA the height was 0.35 ± 0.05 nm and width 7 ± 2 nm, similarly to the results on APTES-mica. The contour length of dsDNA linear pBR322 (4361 bp) molecules was also compared for APTES and ‘‘GM’’ adsorption methods. In the case of APTES mica the contour length was 1480 ± 9 nm (average over 50 molecules) and corresponded with the theoretical value of 1483 nm. In the case of ‘‘GM’’ modifier the contour length was 1525 ± 15 nm, which was 3% longer than in the case of APTES mica. Thus, dsDNA was slightly stretched on ‘‘GM’’. In case of ssDNA the contour length could only be measured for ssDNA adsorbed on ‘‘GM’’ modified HOPG. The contour length of ssDNA M13mp18 on HOPG was 2590 ± 30 nm (average over 25 molecules). It is 5% longer in comparison with theoretical value of dsDNA but 10% shorter in comparison with the contour length of extended molecules of ssDNA M13mp18 in the case of poly-lysine. It means that this deposition technique for ssDNA on modified HOPG does not put ssDNA under significant stress. The contour length of ssDNA M13mp18 corresponds to 0.36 nm/base while in case of poly-lysine it was 0.39 nm/base. It correlates well with recent transient electric birefringence measurements that indicate a spacing of 0.32–0.52 nm/base for ssDNA, depending on sequence. In Fig. 3C and D the deposition of heated Lambda dsDNA similarly as it was done for Fig. 2B and D was repeated. By comparing these two sets of images, one can easily note that in the case of ‘‘GM’’ modified HOPG (Fig. 3C and D), the dsDNA as well as the ssDNA are well visible and in a 2D relaxed state. Also on these images, it is possible to observe two ssDNA strands emanating from a dsDNA strand as indicated by the circles in Fig. 3C and D.

Fig. 3. AFM images of: (A, B) ssDNA M13mp18 and (C, D) heated Lambda dsDNA adsorbed on modified HOPG. The bars represent 250 nm on all images.

Images were collected using a Nanoscope IIIa (Veeco Inc., Woodbury, NY, USA) operated in tapping mode in air. HRC with a nominal tip radius of <10 nm were used and were driven at oscillation frequencies in the range of 150–300 kHz. During imaging, the surface was scanned at a rate of one line per second. Images were simply flattened using the Nanoscope III software and no further image processing was done. The contour length of DNA molecules was measured with Ellipse.

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

[1] J. Adamcik, D.V. Klinov, G. Witz, S.K. Sekatskii, and G. Dietler, Observation of single-stranded DNA on mica and highly oriented pyrolitic graphite by atomic force microscopy, FEBS Lett. 580 (24), 5671–5 (2006)