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2008, 112, 9267–9269 Published on Web 07/11/2008
High-Resolution STM Imaging of Novel Single G4-DNA Molecules Errez Shapir,§ Lior Sagiv,§ Natalia Borovok,⊥ Tatiana Molotski,⊥ Alexander B. Kotlyar,*,⊥,‡ and Danny Porath*,§,† Physical Chemistry Department, The Hebrew UniVersity, Jerusalem 91904, Israel, and Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel AViV UniVersity, Ramat AViV, 69978 Israel ReceiVed: April 22, 2008; ReVised Manuscript ReceiVed: June 13, 2008
The molecular morphology of long G4-DNA wires made by a novel synthetic method was, for the first time, characterized by high-resolution scanning tunneling microscopy (STM). The STM images reveal a periodic structure seen as repeating “bulbs” along the molecules. These bulbs reflect the helix morphology of the wires. The STM measurements were supported by a statistical morphology analysis of the DNA pitch length and apparent height relative to the surface. In the absence of X-ray and NMR data for these wires, the STM measurements provide a unique alternative to characterize the helix morphology. The electrical properties of double-stranded (ds) DNA and its derivative G4-DNA were studied as candidates for nanoelectronics.1–4 As it is widely acceptable, poly(dG)-poly(dC) provides the best conditions for π-overlap among dsDNA sequences.5 Moreover, guanine (G) bases have the lowest ionization potential among DNA bases, promoting charge migration through the G bases of the DNA. Taking it one step further, the G4-DNA,6 which is derived from the poly(dG)poly(dC) and has four parallel G stacks, may be an even better candidate because it is stiffer, made of G bases only, and already showed clear electrostatic polarizability.7 While the structure of short, up to few nanometers, guanine-based tetrads was revealed by X-ray,8 there is no structural measurement of the G4-DNA by X-ray or by NMR, mainly due to the complexity of such a large molecule and its only recent availability. In the absence of such data, STM provides the best direct morphology characterization on the single-molecule level for these molecules. We have recently reported the preparation of long monomolecular G4-DNA6,9 made from long continuous poly(dG)poly(dC) molecules.10–12 These molecules were characterized by various biochemical6 and physical7 techniques. Investigation of these polymers by STM has never been performed. STM is a powerful tool for obtaining both morphological and electrical information on the submolecular level. Imaging of DNA has taken an important place in STM activity since its invention.13–27 Although STM does not provide the resolution of X-ray measurements, which is not yet available for these molecules, it can nevertheless provide important morphological information in real space and on the single-molecule level. Here, we report the characterization of these novel continuous long G4-DNA molecules using STM. The results of highresolution STM images reveal a periodic structure seen as * To whom correspondence should be addressed. E-mail: porath@ chem.ch.huji.ac.il (D.P.);
[email protected] (A.B.K.). § The Hebrew University. ⊥ Tel Aviv University. † Also at the Center for Nanoscience and Nanotechnology, The Hebrew University, Jerusalem 91904, Israel. ‡ Also part of the Tel Aviv University Nanotechnology Center.
10.1021/jp803478f CCC: $40.75
Figure 1. High-resolution STM images of G4-DNA molecules deposited on a gold(111) surface. The scans were performed at Vbias ) 2.8 V and Iset ) 20 pA. (a) Image of long DNA molecules of about 250 nm and one bundle located in the middle of the figure (indicated by black arrows). (b) Image of one of the DNA molecules at higher resolution, where the periodic structure is clearly observed.
repeating bulbs along the molecules, reflecting the helix morphology of the G4-DNA. G4-DNA molecules were deposited from a solution in 2 mM tris-acetate (pH 7.0) onto a highly smoothed gold(111) surface achieved by flame annealing. The G4-DNA molecules were 750 tetrads long and contained 40 phosphothioated G-nucleotides at the 5′ end of the molecule.28 The immobilization of the molecules on a gold substrate29–33 via phosphothioated nucleotides28 is an important advantage, although it does not provide a perfect attachment since DNA molecules are commonly displaced or disassembled by the STM tip during repeating scans. All of the presented measurements were performed with a commercial Omicron LTSTM system in ultrahigh vacuum (UHV) conditions (∼5 × 10-11 mbar) at room temperature (RT), at T ∼ 78 and 4.5 K.11,34 The STM results showed that the imaging quality was temperature-invariable. Figure 1a shows a STM image of 750 tetrads long (∼250 nm) single G4-DNA molecules and a bundle (indicated by arrows) at Vbias ) 2.8 V and Iset ) 20 pA. A higher-resolution image of a selected molecule is shown in Figure 1b, where details of the pitch structure of the DNA can be seen. The underlying gold(111) surface, as well as grain boundaries on it, can be clearly distinguished from the molecules. The high-resolution scans presented in Figure 2 further demonstrate the clear periodic structure of the G4-DNA. The 2008 American Chemical Society
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Letters
Figure 2. Detailed STM images showing clear periodic structure of G4-DNA molecules scanned at Vbias ) 2.8 V and Iset ) 20 pA. Panels (a) and (b) demonstrate the apparent molecule morphology. The height with respect to the surface and the average twist length are shown in the corresponding cross sections in the insets. (c) A selected molecule in a 3D perspective, in which a possible defect is seen and marked by a yellow arrow.
Figure 3. Graphic presentation of the morphological statistics performed on the STM images of G4-DNA. The statistical analysis was carried out on ∼150 different sites on different DNA molecules. (a) The molecule twist length distribution shows a peak at 3.6 nm. (b) The molecule apparent height distribution shows a peak at 1.5 nm.
molecular height obtained from a cross section across the molecule (shown in the inset of Figure 2a and marked by a black line) is ∼1.5 nm. This reduced height compared to the molecule diameter is consistent with standard results of height imaging of DNA molecules on surfaces by both STM and atomic force microscopy. A lower measured height on the surface compared to the molecular diameter as characterized in solutions and crystals is probably due to surface forces and poor electrical transmittivity of the molecules.11,34 Nevertheless, this apparent topographic height is almost twice the height of dsDNA in our previous report,11,34 measured at similar conditions and with the same system, probably due to a combination of the higher molecule stiffness and possible better transverse conductivity than that of poly(dG)-poly(dC). In Figure 2b (top view) and c (3D perspective), the periodic structure is visualized along the molecule, with bulbs corresponding to the helix pitch with an average length of ∼3.5 nm. A corresponding cross section (marked by a black line) in the inset of Figure 2b shows nine peaks related to nine twists of the molecule helix. Some possible structural defect is also observed in the molecule (indicated with a yellow arrow in Figure 2c). Discriminating the features of the gold substrate from those of the DNA molecules is fairly straightforward as they are visually very different. Beyond the visual appearance of the images, there were additional indications for discriminating the DNA molecules from the surrounding surface. First, sometimes when a single DNA molecule was scanned repeatedly, it was finally disassembled due to occasional physical contact with the STM tip or due to the reapplied electric field. These cases showed clearly that the molecule is a distinctive soft material over a harder gold surface. Molecule disassembly and displacement by the STM tip was much more common in the case of dsDNA, as we previously reported.11,34 In the case of the G4-
DNA, these unwanted phenomena were much less common, probably due to the tighter binding of the molecules via the phosphothioated anchoring residues28 and also possibly due to a better conductance of these G-rich molecules. The apparent height of the DNA molecules, that is, the relative height with respect to the gold surface, is changing upon varying the bias voltage below ∼2V, while the morphology of the underlying gold remains constant. This effect shows that the molecule height measured by STM images is the “electrical” height rather than the physical one compared with the metal surface morphology, which is independent of the voltage34 and is emphasized by the fact that the apparent height of the G4-DNA is almost twice the height of the dsDNA. Statistical analysis of the morphological features, presented in Figure 3a, was carried out on ∼150 different segments on different DNA molecules. Figure 3a shows the helix twist length distribution with a peak at 3.6 nm, an averaged length of 3.5 nm, and standard deviation of (0.2 nm. The average value differs by 0.1 nm from the value measured by X-ray using crystalline short (few nanometers) G4-DNA,8 which is within the experimental error. Figure 3b presents the apparent height distribution with a peak at 1.5 nm, average value of 1.5 nm, and standard deviation of (0.2 nm. Optimal images were obtained at bias voltages of 2.5-2.8 V and current sets of 20-80 pA, which are the most suitable conditions for STM imaging of DNA with the measurement system at hand. The statistical data showed no variation under the above scan parameters for all of the temperature range from RT down to ∼4.5 K. We note here that we do not expect the structure of G4-DNA adsorbed on a gold surface in ultrahigh vacuum conditions to be the same as that in solution or that in physiological conditions. The molecular structure is probably distorted to some extent due to both the surface forces applied to the molecule and due to
Letters stripping of most of the water around the molecule except some layers of structured water in UHV conditions. In summary, we report a reproducible single-molecule imaging characterization of novel continuous G4-DNA molecules using STM. High-resolution images of the G4-DNA molecules have been elucidated. Morphological statistics has been made for the molecules, resolving the periodicity (the helix pitch lengths) and the apparent height of the molecules with respect to the surface at the reported vacuum and temperature conditions. The STM characterization provides a direct way of revealing the G4-DNA morphological structure on the singlemolecule level on surfaces, which is not yet available through current structural characterization methods, such as X-ray and NMR. Our study indicates that these novel molecules conform quite closely to standard knowledge about G4-DNA, with small deviation. Acknowledgment. We thank Igor Brodsky for assistance and fruitful discussions. This work was supported by the Israel Academy of Science and Humanities, the German Israel Foundation, and European grants for Future & Emerging Technologies DNA-Based Nanowires (IST-2001-38951) and DNA-based Nanodevices (FP6-029192). References and Notes (1) Porath, D.; Bezryadin, A.; Vries, S. D.; Dekker, C. Nature 2000, 403, 635. (2) Porath, D.; Cuniberti, G.; Di Felice, R. Long-Range Charge Transfer DNA II 2004, 237, 183. (3) Endres, R. G.; Cox, D. L.; Singh, R. R. P. ReV. Mod. Phys. 2004, 76, 195. (4) Ventra, M. D.; Zwolak, M. In DNA Electronics. Encyclopedia of Nanoscience and Nanotechnology; Nalwa, H. S. Ed.; American Scientific Publishers: Valencia, CA, 2003; p 475. (5) De Pablo, P. J.; Moreno-Herrero, F.; Colchero, J.; Go´mez Herrero, J.; Herrero, P.; Baro´, A. M.; Ordejo´n, P.; Soler, J. M.; Artacho, E. Phys. ReV. Lett. 2000, 85, 4992. (6) Kotlyar, A. B.; Borovok, N.; Molotsky, T.; Cohen, H.; Shapir, E.; Porath, D. AdV. Mater. 2005, 17, 1901. (7) Cohen, H.; Sapir, T.; Borovok, N.; Molotsky, T.; Di Felice, R.; Kotlyar, A. B.; Porath, D. Nano Lett. 2007, 7, 981. (8) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668. (9) Borovok, N.; Molotsky, T.; Ghabboun, J.; Porath, D.; Kotlyar, A. B. Anal. Biochem. 2008, 374, 71.
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