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© Copyright 1996 American Chemical Society
APRIL 3, 1996 VOLUME 12, NUMBER 7
Letters Imaging of Single Extended DNA Molecules on Flat (Aminopropyl)triethoxysilane-Mica by Atomic Force Microscopy J. Hu,†,‡ M. Wang,§ H.-U. G. Weier,§ P. Frantz,† W. Kolbe,| D. F. Ogletree,† and M. Salmeron*,† Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720 Received October 16, 1995. In Final Form: January 23, 1996X Long strands of DNA have been deposited, extended, and immobilized on chemically modified mica for study with an atomic force microscope (AFM). By employing a modified “molecular combing” technique and carefully silanized mica, we show that DNA molecules may be distributed uniformly, adsorbed in extended conformations, and bound with great tenacity. When maintained in a buffer solution, these strands remained stable for >5 h. Scanning the surface with high loads applied to an AFM tip induced fragmentation in the strands, but remaining pieces never relinquished their original positions. Further imaging applications, such as after treatment of the DNA by molecular hybridization or DNA synthesis and labeling in situ, are discussed.
Introduction Since the invention of the atomic force microscope (AFM),1,2 imaging of DNA has been one of the most eagerly anticipated applications. Among the advantages of using the atomic force microscope (AFM) to image DNA are the high spatial resolution (5 h. Finally, we discuss encouraging experiments in which biological processes were shown to occur under these conditions. Experimental Section Sample Preparation. The mica substrates were coated with a self-assembled monolayer of (3-aminopropyl)triethoxysilane (APS, United Chemical Technology Co., Bristol, PA). Solutions were prepared by mixing the APS with distilled-deionized (dd) water, concentration 1% by volume, and filtered through a 2 µm cellulose acetate syringe filter. Freshly cleaved mica sheets were immersed in the APS solution for ∼5 min and rinsed thoroughly with dd water afterward. DNA solutions were composed of lambda DNA (48 502 base pairs (bp), from GIBCO/LTI, Gaithersburg, MD), concentration 1 ng/mL in dd water. For study with the AFM, the samples were prepared by first depositing a small drop of DNA solution (about 1 µL) onto a clean glass coverslip. The coverslip was then carefully laid on top of the APS-mica with the solution between the two surfaces. The drop spread immediately as the cover glass and the APS-mica sealed together. After waiting for a few minutes, the cover glass was removed and the APS-mica surface was rinsed with dd water thoroughly and blown dried with clean compressed air or nitrogen. While images were collected with the AFM, the samples were contained in a liquid cell and immersed in a buffer solution known as Tris. This is an aqueous solution of HCl, 50 mM concentration, with an equal concentration of salt (either NaCl, KCl, or MgCl). Typical pH values were in the range of 7-9.5. (13) Detailed results are to be published elsewhere. Briefly, an aqueous solution of APS, 1% by volume, is used here. The container must be very clean. A freshly cleaved mica sheet is immersed into the APS solution for several minutes. APS forms a thin, disordered monolayer ∼5-8 Å thick on the mica surface. The surface of APS-mica prepared in this way is very flat, with a roughness of ∼3 Å over several micrometers. The charge state of the amine group of APS on mica is dependent upon the pH of the solution.
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Langmuir, Vol. 12, No. 7, 1996 1699
Figure 2. 500 × 500 nm AFM images of the same DNA strand: (a, left) under an applied load of 1 nN; (b, right) after high-load scanning in a small range in the middle of the strand. A piece of the strand in this range has been swept away by the tip. Instrumentation. The AFM used in this study is home built,14 with a commercial electronics control system (RHK Technology, Rochester Hills, MI). We used Park-sharpened cantilevers with a force constant of 0.1 N/m. All the images were obtained under normal contact mode. Fluorescence microscopy was performed on a Zeiss Axioskop microscope equipped with a 63×/1.25 N.A. oil immersion lens and multibandpass fluorescence filters (Chroma Technology, Brattleboro, VT).
Results and Discussions Initially, DNA samples were stained with 1 µM YOYO (Molecular Probes, Eugene, OR)11,12 for observation with a fluorescence microscope to determine the distribution and orientation of strands on the APS-mica surface. For this measurement, the glass coverslip remained in place after deposition and elongation of the DNA. The results were indistinguishable from DNA deposited onto silanized glass substrates.12 The strands were highly extended and distributed quite uniformly. No motion of the DNA could be detected by optical microscopy. Since the DNA remained immersed in water between the slide and coverslip, we concluded that the adsorption of DNA on APS-mica was stable in water. It was subsequently found that these adsorbed molecules were stable in many buffer solutions, such as Tris, HEPES,10 2× SSC, phosphate, and borate. In addition, the molecules remained in place after repeated rinsing with dd water. However, in the case of a bare mica substrate, DNA molecules could be seen floating unattached under the microscope. This suggests that they are not strongly adsorbed to bare mica in water. (14) Kolbe, W. F.; Ogletree, D. F.; Salmeron, M. B. Ultramicroscopy 1992, 42-44, 1113. (15) DNA was deposited and extended onto APS-mica substrate and denatured by heating at 95 °C for 2 min. The sample was then overlaid with the solution containing Klenow Fragment of DNA polymerase I, random primers from GIBCO/LTI’s BioPrime DNA Labeling System, and a deoxynucleoside triphosphate mixture containing biotin-dCTP. After incubating at 37 °C overnight, slides were washed in 2× SSC and incubated with avidin-FITC for 30 min at 20 °C. Finally, the slides were washed in two changes of 2× SSC and the sample was observed under a fluorescence microscope. The biotin-labeled DNA which was synthesized during extension of the random primers was visible in the microscope as long and continuous green DNA strands. Control samples treated in the same way, except that the Klenow Fragment of DNA Polymerase I was omitted from the buffer, did not produce DNA strands labeled and visible in the fluorescence microscope. These results indicated that DNA synthesis and replication processes occurred along the extended and adsorbed DNA strands. Detailed results will be published elsewhere.
The DNA samples prepared for AFM experiments were not stained to minimize YOYO background on the surface. Figure 1 shows a typical AFM image of extended lambda DNA on APS-mica. Once again, the strands were distributed uniformly across the surface. The surface density was such that the DNA strands seldom overlapped, yet they were common enough to be found in a typical 5 µm × 5 µm image. Despite collecting these images in the normal contact mode, which may often induce motion in the object being observed, the DNA strands remained securely attached in their original positions. This stability persisted for at least 5 h; mechanical instabilities in our liquid cell limited data collection to this amount of time. As indicated below, measurements of a DNA amplification process suggested that the DNA remained stable for much longer than 5 h. We concluded that the strands are securely attached to the surface at frequent points along their length. This remarkable stability was always present when the load applied to the AFM tip was maintained at 5 nN. These variations, possibly caused by inconsistencies in the tip shape, preclude quantitative analysis at this time. The mechanism of adsorption in this system is not yet understood. One attractive possibility is that the DNA may be negatively charged in aqueous solution at neutral pH.10 Thus, the strands would be electrostatically attracted to positively charged NH2 groups on the APSmica surface. Though the strength of this bond may be modest per segment, it would be enormous per strand. Successful sample preparation depended sensitively upon subtleties of the “molecular combing” technique during extension of the DNA. Occasionally, the spreading
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Figure 3. This 1 × 1 µm image illustrates an unsuitably entangled section of DNA. Here, the spreading process was slowed and misdirected by imperfections in the substrate.
was slow and nonuniform due to steps in the mica surface. This dissimilarity between the surfaces of the mica and coverslip resulted in entanglements in the strands, such as those shown in Figure 3, and may also result in aggregation. If the DNA molecules are bound tenaciously to the APSmica surface, do they retain any of their biological functions? It has been found that some biological processes can still be performed on those extended and adsorbed DNA strands. Molecular hybridization has been carried out by Weier et al. and the results in terms of detection sensitivity and reproducibility are quite exciting.12 More recently, enzyme activity on those DNA strands has also been found and DNA strands could be replicated by using the Klenow Fragment of DNA polymerase I and random oligonucleotide primers.15 It is not yet understood how the interaction between the strongly adsorbed DNA and DNA polymerase occurs, but AFM has made it possible to monitor the biological processes involving DNA in situ at the molecular level. The current problem for AFM imaging in the process of DNA amplification is that the dNTP could also be adsorbed on the APS-mica surface. These additional adsorbates produced a rough background which is responsible for the poorer AFM image shown in Figure 4. However, one can still distinguish that the DNA strands are wider than those in Figures 1-3, which might be the result of amplification. Further experiments are needed to ascertain these potentially exciting results.
Letters
Figure 4. A 3 × 3 µm image showing DNA strands after the amplification process. The surface surrounding the strands was significantly coarsened by the presence of adsorbed dNTP or proteins.
In summary, DNA has been prepared on flat APSmica with a modified “molecular combing” method. DNA strands were distributed uniformly, extended straight, and adsorbed tightly on the substrate. We imaged extended DNA in normal buffer solution by AFM and stable images could be obtained routinely. It was found that those DNA molecules remained stable in buffer solution for >5 h and were suitable for AFM imaging. Molecular hybridization and DNA replication processes on those DNA strands were also discussed. Acknowledgment. This work was supported by the Lawrence Berkeley Laboratory through the Director, Office of Energy Research, Basic Energy Science, Materials Science Division of the U.S. Department of Energy under Contract Number DE-AC03-76SF00098 and through the Director, Office of Health and Environmental Research, of the U.S. Department of Energy under Contract Number DE-AC03-76SF00098. J. Hu acknowledges a grant from the Academia Sinica and the Committee of Science and Technology of Shanghai, People’s Republic of China. LA950874C
