Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces
© to the authors 2009
Received: 9 January 2009
Accepted: 14 May 2009
Published: 31 May 2009
To study enzyme–DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I.
Advances in single-molecule techniques make it possible to explore new phenomena and unravel novel mechanisms in biology that were largely inaccessible by traditional bulk measurements . For example, studies of DNA–protein interaction at single molecular level could characterize the distributions of molecular properties and observe the temporal evolution of complicated reaction pathways . It is generally understood that single-molecule measurements require adsorption and fixation of single DNA molecules on a solid support surface [1, 3] before the protein motion along the DNA can be tracked. Among the many kinds of substrate surfaces, mica is ideal because of its atomic smoothness. Since newly cleaved mica is negatively charged at basic pH , an advisable surface modification is critical to bind the negatively charged phosphate backbone of DNA. Typically, poly-l-lysine [5, 6], silane [7, 8], and divalent cations, such as Ni2+ and Mg2+, have been used to provide positively charged sites and/or hydrophobic surfaces for enhancing the interactions between DNA and surfaces [4, 9, 10]. However, these modification methods usually compromise the inherent surface roughness of mica, making it more difficult to gain structural insight into biomolecules with nanometer resolution. Also such modified surfaces are not well suited for dynamic measurements of protein or DNA molecules, because the entire DNA molecule is often fixed tightly on the surface, leading to little or tardy response of the molecule to environmental changes.
To fix DNA on a surface for investigation into its interaction with other reactants, one strategy is to modify the terminal of the DNA strands, so that they specifically bind to surfaces [11–13]. For instance, van Oijen et al. used biotin–avidin system to fix only one end and allow the rest of the single DNA molecule to interact with exonuclease . Medalia et al. demonstrated a method that anchors two ends of a DNA fragment with a thiol group on a gold film-modified mica surface . Recently, a novel strategy named “protein-assisted DNA immobilization” was proposed by Dukkipati et al. in which DNA binding proteins such as restriction enzymes or RNA polymerases are used as attachment points to adsorb DNA on surfaces . Although this method can maintain the biological activity of the immobilized DNA molecules, it is not suitable for higher resolution imaging at nanometer scale by atomic force microscopy (AFM), because hydrophobic polymethylmethacrylate (PMMA) surfaces have to absorb proteins.
We are working on single-molecule enzymatic reactions on mica surfaces by controlled dipping of a nonspecific endonuclease over the DNA molecules based on nanomanipulation . To simultaneously realize the goals of obtaining structural insights into biomolecules with nanometer resolution and providing an appropriate condition for their biological processes, we investigated enzymatic reactions (DNase I) at single DNA molecules attached and immobilized on mica surfaces functioned by gold nanoparticles (GNPs), 1.4 nm-diameter nanoparticles (Nanogold). We demonstrate that Nanogold-modified mica surfaces (Nanogold-mica) have less effect on the length and topography of DNA molecules and provide a suitable environment for higher efficiency of enzymatic reactions on DNA.
Materials and Methods
The original DNA solutions (Shanghai Sangon Biological Engineering Technology and Services Co., Ltd) were diluted to final concentrations of 1 ng/μL for λ DNA and 0.1 ng/μL for pBR322, in TE buffer (10 mM TE–HCl, pH 8.0). Nanogold-mica was produced by treating freshly cleaved mica with 1–50 fM Nanogold (Nanoprobes, Stony Brook, NY) in water for 1 min. After being dried with nitrogen gas, the “spin-stretching” technique was used to stretch and fix DNA . Briefly, 2–5 μL DNA was put on a Nanogold-mica, which was adhered firmly on a centrifuge. The spin speed was limited to <3,000 rpm to extend DNA for 30 s. Samples were washed twice with 10 μL deionized water and dried for imaging.
AFM imaging was conducted using the tapping mode of a MultiMode Scanning Probe microscope (NanoScope IIIa, Digital Instruments, Santa Barbara, CA) with a J Scanner. Noncontact cantilevers (NSC11, MikroMasch) with a resonance frequency of ~300 kHz and a spring constant of ~40 Nm−1were used for imaging at room temperature (in an ambient situation). All AFM images were flattened and analyzed with the microscope’s software system. The contour lengths of single DNA molecules and percentage of DNA occupied on surfaces were determined using METAMORPH software (MDS, Inc.) (see supporting information on the method of calculating DNA length and coverage, supplementary material 1).
For enzymatic digestion of DNA molecules, Dip-Pen Nanolithography (DPN) [16, 18–20] was used to deposit DNase I on DNA. Briefly, an AFM tip coated with 0.01–0.05 unit/μL DNase I (Sigma) in 20 mM Tris–HCl, pH 8.3, 2 mM MgCl2, and 2 mM CaCl2 was mounted on the sample stage. After the first DNA image was obtained by tapping mode, lift mode was turned on to move the AFM tip closer to the surface by setting a negative lift height value. The tip remained for a moment once it touched the surface to induce a meniscus between the tip and the surface. Then, the first image was scanned again with tapping mode but this time by depositing DNase I on the surface and the DNA. Afterwards, several images were recorded in situ to observe the process of DNA digestion. The digestion experiments were conducted in a relative humidity of 30–40% and a temperature of 20–25 °C.
Results and Discussion
As shown in Fig. 1b 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 demonstrated that we are able to facilely deposit and anchor DNA molecules on a mica surface using Nanogold for single-molecule enzymatic reactions. The immobilization of DNA on Nanogold-modified surfaces does not require time-consuming steps, and the fixed DNA strands on the surface can easily be observed on AFM images. Because the Nanogold distribution largely determines the interaction forces between mica and the adsorbed DNA molecules, we could minimize any possible influence of the surface on the native properties of DNA molecules by adjusting the concentrations of nanoparticles, thus providing conditions in which distinct conformations of DNA molecules and their interactions with proteins or other materials can be studied better. By using Dip-Pen Nanolithography to dip DNase I over DNA molecules, we have realized to digest single DNA molecules with higher efficiency. Further research toward more careful control over the deposited density of the Nanogold on surfaces for fixing DNA in solution and probe the structure-related properties of DNA with various kinds of restriction endonucleases needs to be conducted. Some of this research is currently under way in our research group.
This work was supported by grants from NSFC (10675160, 10604061, and 10874198).
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