Nanogold is generally used as a contrast agent in electron microscopy [21
]. In our experiments, we utilize the unique properties of the positively charged Nanogold to act as cross-linker between negatively charged DNA and mica through electrostatic interactions (Fig. a). We expect that most parts of DNA are free except for the binding sites to Nanogold. Due to the fact that only bare mica is used and no other additional surface modification is needed, the inherent surface properties of mica such as its atomic flatness and hydrophilicity are less affected. So the features of DNA can be clearly observed, and a suitable surface for observing the biological activities of proteins can be provided.
aSchematic showing of the Nanogold-modified mica and the anchored DNA on it (not drawn to scale),bAFM topography image of Nanogold on a mica surface, andcThe corresponding cross-section height profile of Nanogold
As shown in Fig. b and c, after the modification process, the Nanogold, 1.4 nm in height, is randomly dispersed on the mica surface. The roughness of the mica surface is changed a little by the sparse distribution of small size nanoparticles. The root mean square (RMS) roughness measured on the 1.75 μm × 1.75 μm area of the mica surface was ~0.06 nm. Although there is a slight increase in this value compared with a freshly cleaved mica surface of ~0.05 nm, it is sufficient for imaging DNA and studying the interaction between protein and DNA.
We have successfully deposited and immobilized DNA molecules in the presence of Nanogold. In principle, a reasonable number of binding events are controlled by varying the nanoparticles’ coverage on the surfaces. An increase in Nanogold concentration increases the attachment points on the surface, thus leading to more DNA binding. Figure shows the results of λ DNA attachment to a modified surface at two different Nanogold concentrations. In the case of 50 and 5 fM Nanogold, the coverage of DNA fixed on Nanogold-mica is about 4% (Fig. a) and 1% (Fig. b) respectively. Depending on the application, a different coverage of DNA attachment can be obtained. However, a higher density of Nanogold would influence the topography of DNA, thus it is important to control the numbers of Nanogold on mica surface to achieve a better DNA topography. In Fig. b, there are a few nanoparticles that are used to attach lambda DNA molecules on the surface, and the lower DNA molecule is anchored only by a single Nanogold. From the cross-section profile of Fig. c as shown in Fig. d, the measured height of the binding site is 1.8 nm (arrow 1), equaling the value of DNA height of 0.4 nm (the measured height of most parts of DNA, arrow 2), plus a Nanogold height of 1.4 nm (arrow 3). In addition, there is the measured height of 0.8 nm (three thin arrows in Fig. c) along DNA strands, implying other structures of DNA existing on the surface.
Figure 2 Typical AFM images of lambda DNA anchored on Nanogold-mica modified witha50 fM andb5 fM Nanogold. Height bar = 5 nmcAn enlarged image from the mini square in Fig. b. Height bar = 2 nmdA height profile of DNA indicated by alinein Fig. (more ...)
We have also explored the general applicability of Nanogold to deposit circular and linear DNA on mica. Circular pBR322 DNA and Pst1
linearized pBR322 were chosen for this purpose. It has been reported that the enzyme sometimes shows limited catalytic activity on overstretched DNA molecules. Although it is possible to avoid overstretching by reducing the hydrophobic effects during the DNA-stretching processes [22
], the problem of controlling this effect persists. However, in our experiments, DNA molecules are easily attached but not overstretched. As shown in Fig. , the measured lengths of DNA range from 1.31 to 1.48 μm regardless of linear or circular molecules, which is very close to the actual length, 1.48 μm. The preserved conformation of DNA would be a potential advantage for reactions of DNA with other molecules like proteins and enzymes.
AFM images of DNA anchored on Nanogold-mica surfaces.aStretchedPst1linearized pBR322.bCircular pBR322
After being able to reproducibly deposit linear and circular DNA molecules on mica without overstretching them, it would be very interesting to explore whether DNA molecules attached on Nanogold-mica are beneficial for the investigation into enzymatic reactions along a single DNA molecule. To this end, a digestion reaction with DNase I was carried out. DNase I is a paradigm endonuclease used routinely for nonspecific cleavage of DNA in molecular biology. Figure shows the process of the enzymatic reaction. The uniform linear DNA (Fig. a) was digested into several fragments immediately (Fig. b) after DNase I ink (bright spots in image) was transferred from the coated tip to the surface and DNA. The size of spots changed along with the time passed. About half an hour later, the volume of the ink spots decreased greatly (Fig. c). To observe DNA clearly, the sample was imaged again after 10 h. All bright spots and most parts of DNA disappeared, but tracks of DNA still remained (Fig. d). This phenomenon is interesting, its mechanism however is unclear so far. We think the disappearance of ink (Fig. b–d) may be caused by the tip’s effects, such as tip-induced diffusion and/or adsorption, during scanning processes. Other factors, such as liquid evaporation and liquid diffusion may also play a rule. To exclude any chance that the observed gaps could have been caused by mechanical force applied by the AFM tip, control experiments with denatured enzyme were performed, and no such digestion phenomenon occurred. The results imply that the flat, hydrophilic Nanogold-mica surface is suitable for the detection of enzymatic digestions of DNA by AFM. We note that no additional sample washing steps were needed; therefore, this technique not only completely eliminates any possible artifacts caused by the water flow, but also has the potential to be developed into a method for recording digestion reactions in a time-lapse manner. It should be noted that although the cleavage of DNA can be observed on other modified surfaces, such as APTES-mica [16
] and Ni-mica [23
], using Nanogold-mica facilitates the detection of small gaps in the DNA due to the relatively free state of the molecule. Most of the DNA has weak interaction with the surface except at the points that are anchored by Nanogold. Once the phosphodiester linkages are broken, the ends of the DNA fragments have a tendency to adjust their positions because of their entropic property, so a larger gap appears. Additionally, the modified surface is flat, providing a unique platform to probe the topography of DNA. Moreover, the entire smooth surface is hydrophilic because of the hydrophilic mica surface and the water soluble Nanogold. The flat, hydrophilic surface facilitates ink and small DNA fragments to diffuse on the substrate, leading to an enlarged gap and a clear view field. So a digestion reaction of DNA can be probed clearly, even without washing steps.
AFM images of DNA reaction of digestion by DNase I. Height scales = 8 nm except for (a).aDNA topography before digestion. Height scale = 2 nm.bDNA fragments just after a DPN process.cDNA fragments after DPN 0.5 h.dTraces of DNA after DPN 10 h