Preparation of samples of DNA molecules for AFM measurements

M. N. Savvateev
 NT-MDT, Moscow, Russia.

Atomic Force Microscopy (AFM) makes it possible to obtain images with the resolution of several nanometers [1-3] and it has been actively employed for the study of structure specifics of biological macromolecules (protein, DNA) these days. In addition to studying dry samples, AFM allows study of molecules in buffer solutions.
The process of imaging by means of AFM requires the objects under investigation to be fixed to some surface. Crystal mica, which has atomic level smooth surface, is typically used as substrate to study DNA molecules. There are several methods of fixation of negatively charged DNA molecules on mica, whose surface also has a negative charge in water. This article presents a review of those fixation techniques. The AFM images, presented in this article as illustrations, have been obtained by means of Solver and Ntegra systems using semi-contact techniques in air and liquid.

1. Addition of ions of two-valent metals.

The simplest technique is the addition of ions of two-valent metals to the buffer. These ions play the role of bridges between the negatively charged surface of mica and the DNA molecule. According to work [4], ions of nickel, cobalt and zinc are the most suitable for fixation of DNA molecules on mica as they provide binding with the surface strong enough for measurements in liquid.
The sample preparation procedure is as follows: The DNA solution is dissolved in buffer containing 5mĚ of HEPES and 1-5 mĚ of NiCl2 so that its final concentration becomes approximately equal to 1 microgram per ml. Next, a drop of this solution is applied on the mica substrate and it is incubated for 5 minutes. Measurements can be then performed either in the buffer (Fig. 1a) or, after washing it in water and drying, in air (Fig. 1b).

a	b
Figure 1. AFM images of a circular plasmid DNA (pEGFP, 3.4 kb). This sample is prepared using ions of nickel. The image (a) is obtained by a semi-contact technique in buffer. The image (b) is obtained by a semi-contact technique in air.

The binding by means of ions of magnesium is weaker and, therefore, is not used for DNA measurements directly in the buffer solution. However freshly cleaved mica provides better bounding between DNA molecules and its surface after being kept in water immediately before applying the solution on it [5]. The bond strength can be assessed by the shape of the molecules: the relaxed shape (Fig. 2a) implies the presence of two-dimensional diffusion over the mica surface, and therefore it implies that the interaction is weak. While the twisted shape (Fig. 2b) indicates that the molecules are captured on the surface once they come in contact with it [5].

a	b
Figure 2. DNA molecules of the same solution applied on a freshly cleaved mica (a) and a mica sample after having it kept in water (b). Both images are obtained by a semi-contact technique in air.

2. Modification of mica surface using 3-aminopropyltriethoxysilane (APTES) [6].

A film of APTES (3-aminopropyltriethoxysilane), which has a positive charge in water, is formed on the surface of mica. The procedure is following: slices of freshly cleaved mica are placed in an desiccator in the presence of APTES and DIPEA (N,N-Disopropylethylamine) and are incubated in an argon atmosphere for 2 hours at room temperature. The DNA solution, without ions of metals in the buffer, is applied on the prepared therefore substrate. These samples are suitable for measurements both in air and water. Unlike the samples prepared by means of ions of metals, the DNA molecules of the samples prepared using this technique are more twisted (Fig. 3).

Figure 3. Plasmid DNA on mica with a film of APTES on the surface. This image is obtained by a semi-contact technique in water..

3. Modification of mica surface using poly-L-lysine.

A drop of 0.01% solution of poly-L-lysine with molecular weight 30000-70000 is applied on the surface of freshly cleaved mica. It is then incubated for 15 minutes in a wet chamber, washed and dried. The DNA molecules are added and measured either directly in the buffer, without drying the sample, or, after washing and drying it, in air. This sample preparation technique provides a firm bond, however it is characterized with heavy condensation of molecules – most of the molecules form condensates resembling flowers and rods (Fig. 5).

Figure 5. Plasmid DNA on mica treated with poly-L-lysine. This image is obtained by a semi-contact technique in air.

As seen from the figures, the shape of DNA molecules on the surface of mica essentially depends on the sample preparation technique. Applying different preparation procedures to the same solution, it is possible to obtain molecules that are both completely relaxed and heavily condensed. This should be noted when performing research, and the selection of the preparation procedure should be motivated by the conditions of the scientific problem. For example, salts of two-valent metals are more suitable to perform measurements on molecules that need to be straightened (in particular, when studying DNA-protein complexes and performing DNA mapping). While mica substrates modified with 3-aminopropyltriethoxysilane or poly-L-lysine are more appropriate for the study of DNA molecules condensation processes. 

References

1. Maliuchenko N.V., Tonevitsky A.G., Savvateev M.N. et al, Biofizika, v. 48, 5, p. 830-836, 2003.
2. Savvateev, M.N., Kozlovskaya, N.V., Moisenovich, M.M. et al, AIP Conference Proceedings, V. 696, p. 428, 2003.
3. Savvateev, M.N., application note “AFM study of macromolecules' structure and organization”, NT-MDT web site (www.ntmdt.ru).
4. Hansma, H.G., Laney, D.E., Biophys J., 70(4), pp. 1933-1939, 1996.
5. Rivetti, C., Guthold, M. and Bustamante, C., J. Mol. Biol. V. 264, pp. 919–932, 1996.
6. Lyubchenko, Y.L., Gall, A.A., Shlyakhtenko, L.S., Harrington, R.E., Oden, P.I., Jacobs, B.L. and Lindsay, S.M., J. Biomolec. Struct. Dyn., V. 9, pp. 589-606, 1992.