Evaluation of the Radial Deformability of Poly(dG)−Poly(dC) DNA and

Jan 22, 2010 - In fact, it has been suggested that the radial deformability of ..... (unit: volts) on the height measurement has been incorporated, al...
0 downloads 0 Views 5MB Size
Download PDF
pubs.acs.org/Langmuir © 2010 American Chemical Society

)

Evaluation of the Radial Deformability of Poly(dG)-Poly(dC) DNA and G4-DNA Using Vibrating Scanning Polarization Force Microscopy Huabin Wang,†,^ Jiwei Lin,‡ Chunmei Wang,‡ Xuehua Zhang,§ Hongjie An,‡ Xingfei Zhou,*, Jielin Sun,*,‡ and Jun Hu†,‡

)

† Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China, ‡Bio-X Life Science Research Center, Shanghai Jiao Tong University, Shanghai 200030, China, §Department of Chemical Engineering, University of Melbourne, Parkville, Victoria 3010, Australia, and Department of Physics, Ningbo University, Ningbo 315211, China. ^ Current address: Department of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia.

Received November 14, 2009. Revised Manuscript Received January 10, 2010 Poly(dG)-poly(dC) DNA and G4-DNA are two types of synthetic molecules that are regarded as promising candidates for molecular nanodevices. In this work, vibrating scanning polarization force microscopy (VSPFM) was employed to study the radial deformability of these two molecules coadsorbed on a Ni2þ-modified mica surface. The values of the radial compressive elastic modulus of these two types of molecules were found to be similar (∼5-10 MPa) when the external loading force was between ∼0.04 and ∼0.12 nN. However, G4-DNA molecules possessed higher stiffness than poly(dG)-poly(dC) DNA (∼20-40 vs ∼10-20 MPa) when the loading force was larger than ∼0.12 nN. The results will aid us in understanding these molecule’s mechanical performances.

Introduction The DNA molecule has been widely accepted as a smart material for nanotechnology due to its appealing chemical and physical properties.1 In recent years, in addition to DNA-based molecular nanowires, arrays, and objects, a number of DNAbased nanomechanical devices have been made.1,2 Poly(dG)poly(dC) DNA3 and G4-DNA4 (a quadruple helical motif of stacked guanine tetrads) are two novel DNA derivatives with a similar nominal diameter of ∼2 nm and are regarded as promising candidates for molecular nanodevices.5 There has been recent emergence of work focusing on the properties of these two kinds of molecules: their morphology and polarizability have been investigated using atomic force microscopy (AFM),4,6 scanning tunneling microscopy (STM),7,8 and electrostatic force microscopy.5 It is expected that insights into the mechanical properties of these molecules under external loads will aid in understanding the final structural performance and mechanical-related electronic properties of the molecular devices. In fact, it has been suggested that the radial deformability of G4-DNA and dsDNA molecules might influence their electronic properties.5 On the other hand, poly(dG)-poly(dC) and G-quadruplex-forming sequences are also believed to be involved in many important *To whom correspondence should be addressed. E-mail: zhouxingfei@ hotmail.com (X.Z.), [email protected] (J.S.).

(1) Seeman, N. C. Nature 2003, 421, 427–431. (2) Yan, H.; Zhang, X. P.; Shen, Z. Y.; Seeman, N. C. Nature 2002, 415, 62–65. (3) Kotlyar, A. B.; Borovok, N.; Molotsky, T.; Fadeev, L.; Gozin, M. Nucleic Acids Res. 2005, 33, 525–535. (4) Kotlyar, A. B.; Borovok, N.; Molotski, T.; Cohen, H.; Shapir, E.; Porath, D. Adv. Mater. 2005, 17, 1901–1905. (5) Cohen, H.; Sapir, T.; Borovok, N.; Molotsky, T.; Felice, D. R.; Kotlyar, A. B.; Porath, D. Nano Lett. 2007, 7, 981–986. (6) Klinov, D.; Dwir, B.; Kapon, E.; Borovok, N.; Molotsky, T.; Kotlyar, A. Nanotechnology 2007, 18, 225102. (7) Shapir, E.; Cohen, H.; Borovok, N.; Kotlyar, A. B.; Porath, D. J. Phys. Chem. B 2006, 110, 4430–4433. (8) Shapir, E.; Sagiv, L.; Borovok, N.; Molotski, T.; Kotlyar, A. B.; Porath, D. J. Phys. Chem. B 2008, 112, 9267–9269. (9) Kohwi, S. T.; Kohwi, Y. Nucleic Acids Res. 1991, 19, 4267–4271.

Langmuir 2010, 26(10), 7523–7528

biological processes, for instance, in gene regulation.9,10 Many studies have shown that the local flexibility of the DNA molecule is closely related to its biological activity, in processes such as DNA-protein recognition.11,12 Obviously, knowledge of the local deformability of poly(dG)-poly(dC) DNA and G4-DNA is helpful for developing a deeper understanding of their biological functions.13 Unfortunately, information regarding the deformability of poly(dG)-poly(dC) DNA and G4-DNA molecules is extremely limited due to the lack of suitable experimental techniques and/or the only recent availability of these molecules. AFM is a powerful tool for obtaining both morphological and mechanical information on the single molecule level.14-19 In a very recent study, based on the heights measured from ac-mode AFM (similar to tapping mode AFM (TM-AFM)) images at different amplitude set-point values, individual single-walled and double-walled carbon nanotubes have been identified.20 As we have demonstrated in our previous studies, however, this technique is not suitable for precise investigation of the deformability of soft materials like DNA and proteins because these molecules are easily deformed by the AFM tip during the initial imaging (10) Kan, Z. Y.; Yao, Y.; Wang, P.; Li, X. H.; Hao, Y. H.; Tan, Z. Angew. Chem., Int. Ed. 2006, 45, 1629–1632. (11) Fujii, S.; Kono, H.; Takenaka, S.; Go, N.; Sarai, A. Nucleic Acids Res. 2007, 35, 6063–6074. (12) Kalodimos, C. G.; Bonvin, A. M. J. J.; Salinas, R. K.; Wechselberger, R.; Boelens, R.; Kaptein, R. EMBO J. 2002, 21, 2866–2876. (13) Zhou, X. F.; Sun, J. L.; An, H. J.; Guo, Y. C.; Fang, H. P.; Su, C.; Xiao, X. D.; Huang, W. H.; Li, M. Q.; Shen, W. Q.; Hu, J. Phys. Rev. E 2005, 71, 062901. (14) Alessandrini, A.; Facci, P. Meas. Sci. Technol. 2005, 16, R65–R92. (15) Wang, H. B.; An, H. J.; Zhang, F.; Zhang, Z. X.; Ye, M.; Xiu, P.; Zhang, Y.; Hu, J. J. Vac. Sci. Technol. B 2008, 26, L41–L44. (16) M€uller, D. J.; Dufr^ene, Y. F. Nat. Nanotechnol. 2008, 3, 261–269. (17) Goodman, R. P.; Schaap, A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Science 2005, 310, 1661–1665. (18) Li, H. B.; Zhang, W. K.; Zhang, X.; Shen, J. C.; Liu, B. B.; Gao, C. X.; Zou, G. T. Macromol. Rapid Commun. 1998, 19, 609–611. (19) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346–349. (20) Deborde, T.; Joiner, J. C.; Leyden, M. R.; Minot, E. D. Nano Lett. 2008, 8, 3568–3571.

Published on Web 01/22/2010

DOI: 10.1021/la904329q

7523

Article

Wang et al.

process, even before the compression measurement can be carried out.21,22 Recently, we developed a technique termed vibrating scanning polarization force microscopy (VSPFM),13,21 which is derived from scanning polarization force microscopy (SPFM).23,24 VSPFM is featured for its stable performance in both the noncontact and tapping regimes and has proven to be a promising tool for the investigation of the deformability of individual soft molecules.13,25,26 In this report, we have further improved this technique and applied it to quantitatively evaluate the radial deformability of poly(dG)-poly(dC) DNA and G4-DNA molecules at subnanometer resolution. We succeeded in directly measuring the difference in the radial compression elasticity of the two types of DNA molecules.

Experimental Section Sample Preparation. Poly(dG)-poly(dC) DNA and G4DNA were synthesized based on the protocols described in two recent publications.3,4 In the substrate preparation, a drop of 10 μL Ni(NO3)2 solution (10 mM) was deposited onto a piece of Parafilm M laboratory sealing film (American National Can Co.), and then a freshly cleaved mica surface (∼1  1 cm2) was covered on the droplet and incubated for 3 min before the mica was rigorously rinsed with Millipore water (18.2 MΩ).27 The reasons to choose Ni2þ-treated mica as the substrate are described as follows. Mica is a commonly used substrate for imaging of DNA molecules.28,29 However, freshly cleaved mica is negatively charged, which is not suitable to attach negatively charged DNA molecules. Two methods are widely used to attach DNA molecules onto mica. In the first method, small cations are mixed together with DNA solution, while in the second method mica is pretreated with small cations.30,31 The first method is not suitable for our present study since it has been reported that cations may influence the structure of DNA molecules.32,33 Thus, we adopted the second one and modified the mica surface using Ni2þ ions before the attachment of DNA molecules.30 The binding of DNA molecules onto the mica surface can be achieved with the help of the Ni2þ ions that adsorb into the “cavities” at the mica surface and bridge the negative charges of DNA and the negative mica sites.30 This method can minimize the effect of cations on the mechanical properties of DNA molecules. Poly(dG)-poly(dC) DNA and G4-DNA samples were prepared for AFM scanning by simultaneous deposition of 5 ng/μL of poly(dG)-poly(dC) and 5 ng/μL of G4-DNA onto the Ni2þmodified mica surface. The droplet was allowed to incubate for 30 s and gently blotted with a piece of filter paper. A drop of 20 μL Milli-Q water was then deposited onto the sample and the droplet blotted immediately with a piece of filter paper. This rinse step is (21) Li, X. J.; Sun, J. L.; Zhou, X. F.; Li, G.; He, P. G.; Fang, Y. Z.; Li, M. Q.; Hu, J. J. Vac. Sci. Technol. B 2003, 21, 1070–1073. (22) Wang, H. B.; Zhou, X. F.; An, H. J.; Guo, Y. C.; Sun, J. L.; Zhang, Y.; Hu, J. Chin. Phys. Lett. 2007, 24, 644–647. (23) Hu, J.; Xiao, X. D.; Ogletree, D. F.; Salmeron, M. Science 1995, 268, 267– 269. (24) Hu, J.; Xiao, X. D.; Salmeron, M. Appl. Phys. Lett. 1995, 67, 476–478. (25) Zhou, X. F.; Xu, H.; Fan, C. H.; Sun, J. L.; Zhang, Y.; Li, M. Q.; Shen, W. Q.; Hu, J. Chem. Lett. 2005, 34, 1488–1489. (26) Wang, H. B.; Zhou, X. F.; An, H. J.; Sun, J. L.; Zhang, Y.; Hu, J. J. Nanosci. Nanotechnol. 2008, 8, 3864–3867. (27) An, H. J.; Guo, Y. C.; Zhang, X. D.; Zhang, Y.; Hu, J. J. Nanosci. Nanotechnol. 2005, 5, 1656–1659. (28) Fan, F. F.; Bard, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14222–14227. (29) Liu, Z. G.; Li, Z.; Zhou, H. L.; Wei, G.; Song, Y. H.; Wang, L. Microsc. Res. Tech. 2005, 66, 179–185. (30) Pastre, D.; Pietrement, O.; Fusil, S.; Landousy, F.; Jeusset, J.; David, M. O.; Hamon, L.; Le Cam, E.; Zozime, A. Biophys. J. 2003, 85, 2507–2518. (31) Rivetti, C.; Guthold, M.; Bustamante, C. J. Mol. Biol. 1996, 264, 919–932. (32) Duguid, J.; Bloomfield, V. A.; Benevides, A. J.; Thomas, G. J., Jr. Biophys. J. 1993, 65, 1916–1928. (33) Sun, X. G.; Cao, E. H.; Zhang, X. Y.; Liu, D. G.; Bai, C. L. Inorg. Chem. Commun. 2002, 5, 181–186.

7524 DOI: 10.1021/la904329q

crucial for preparing a highly clean sample34 that is required for a precise compression elasticity measurement on the single molecule level. Finally, samples were dried with fresh airflow prior to use. Instrument. The VSPFM was a modified version of a Nanoscope IIIa Multimode SPM system (Veeco, Santa Barbara, CA). It was developed by the combination of SPFM and TMAFM.13,21,24 The basic principle of using VSPFM for compression of individual molecules has been described in our recent work.13 Briefly, in VSPFM the system is initially operated in TMAFM, and then a bias voltage (ac, alternating current, ∼4-6 V) is applied between the conductive tip and the sample to induce an electric polarization field. Because electrostatic force is longrange,35 by adjusting the vibration amplitude set-point (Asp) value stepwise, the tip-sample interactions can be regulated over a wide range, i.e., from the noncontact regime to the tapping regime. In the compression of a target molecule using VSPFM, at a given Asp value (Asp(j)) the molecule’s height (Dj) is the addition of the molecule’s apparent height (Dj-app) and the tip-to-substrate distance. A series of heights of the molecule are obtained by gradually changing the Asp values. The molecule’s deformation can be calculated from the heights of the molecule at different Asp values. The force exerted on the molecule in the region investigated is dominated by the repulsive tip-molecule interaction. In this region the normal force is approximately proportional to Dj-appKc/Q, where Kc and Q denote the spring constant and quality factor of the cantilever, respectively.13,26 The absolute force calibration is obtained by using the same tip that has been used in the VSPFM experiment for pressing a reference cantilever and detecting its deflection at different Asp values.13,36 From the deformation of the molecule and the loading force exerted on it, detailed information on the deformability of the molecule can be deduced. To minimize the drift of the instrument during the experiment, the VSPFM system was installed in a specially designed chamber in which humidity, temperature, and noise level could be wellcontrolled. An E scanner was used, and the z-limit was also minimized (500 nm) to achieve high resolution. Conductive NSC35/Pt-Ti (MikroMasch Co., Russia) rectangular cantilevers were used with a typical force constant of 4.5 N/m and resonant frequency of 150 kHz. The tip radii were estimated to be ∼25 nm by scanning gold nanoparticles (1.4 nm in diameter, Nanoprobes, 95 Horse Block Road, Yaphank, NY) using TM-AFM.37 The spring constants of all cantilevers used were calibrated through the thermal tune function included in Nanoscope v7.20 software on a Nanosope V microscope (Veeco, Santa Barbara, CA). All operations were performed in air under relative humidity of ∼30% at a temperature of ∼25 C.

Results and Discussion In AFM experiments, factors such as temperature and humidity can affect experimental results. In addition, the radius of AFM tip can vary significantly from one tip to another, and usually the tip geometry is not accurately calibrated.37,38 To minimize the extent of these effects on our experiments, we prepared both poly(dG)-poly(dC) DNA and G4-DNA on the same Ni2þ-treated mica surface and characterized them using the same tip in the same operation process. The Ni2þ-treated mica was very smooth with an Rrms value of 0.03-0.05 nm in a scan size of 500  500 nm2. As shown in Figure 1, two different kinds of molecules can be clearly identified in the TM-AFM topography image, where the G4-DNA molecules appear brighter and shorter. The cross (34) Wang, H. B.; Zhang, L. J.; Zhang, F.; An, H. J.; Chen, S. M.; Li, H.; Wang, P.; Wang, X. Y.; Wang, Y.; Yang, H. J. Surf. Rev. Lett. 2007, 14, 1121–1128. (35) Salmeron, M. Oil Gas Sci. Technol. 2001, 56, 63–75. (36) Su, C. M.; Huang, L.; Kjoller, K. Ultramicroscopy 2004, 100, 233–239. (37) Xu, S.; Arnsdorf, M. F. J. Microsc. 1996, 187, 43–53. (38) Liou, J. W.; Mulet, X.; Klug, D. R. Biochemistry 2002, 41, 8535–8539.

Langmuir 2010, 26(10), 7523–7528

Wang et al.

Article

Figure 1. TM-AFM topography images: (a) large-area views of G4-DNA (made of ∼4000 base poly(dG)) and poly(dG)-poly(dC) DNA (∼4000 base pairs) molecules coadsorbed on a Ni2þ-treated mica surface; (b) image of selected G4-DNA and poly(dG)-poly(dC) DNA at higher resolution. The inset shows the height profile along the green line. The scan sizes are 2  2 μm2 in (a) and 650  650 nm2 in (b).

section also clearly shows the height difference of these two types of molecules. The average apparent height of poly(dG)-poly(dC) DNA (∼0.7 nm) is about half that of G4-DNA (∼1.4 nm). In our experiments we also compared the morphology of λ-phage DNA and poly(dG)-poly(dC) DNA deposited on a Ni2þ-treated mica surface and found no observable difference between these two different dsDNA molecules (data not shown). In the literature, Kotlyar et al. showed that the average apparent heights of poly(dG)-poly(dC) DNA and G4-DNA were ∼0.8 and ∼1.6 nm, respectively.4 Later, Cohen et al. reported that the average apparent height of λ-phage DNA and G4-DNA were ∼0.6 and ∼1.2 nm, respectively.5 Thus, the morphologies of poly(dG)-poly(dC) DNA and G4-DNA in the present study are highly consistent with those previously reported. In order to precisely compare the radial compression deformability of these two similar delicate molecules, we extended the VSPFM technique to compress two different molecules in the same operation process. As shown in Figure 2a, the operation is briefly outlined as follows. To begin the test, TM-AFM was used to locate the target molecule segments in a small scan size, and then the segments were imaged with minimal force by adjusting Asp values. Following this, a proper ac voltage was applied between the conductive AFM tip and the substrate to introduce a polarization force by which the tip could be lifted to gently touch the top of the G4-DNA segment, as shown in stage (1). The tip was then lowered by carefully reducing the Asp value stepwise to compress the G4-DNA segment gradually, and at a certain Asp value (i.e., after the ith step of compression) the tip gently touched the top of poly(dG)-poly(dC) DNA segment, as shown in stage (2). The methods to judge the state of the tip touching the top of the target location and the substrate have been detailed in our previous work.13,21 It is important to note that from this stage the poly(dG)-poly(dC) DNA segment would be compressed in following steps, together with the G4-DNA segment. By further reducing the Asp value, the tip finally touched the substrate, as shown in stage (3). In the experiment, a series of height images of G4-DNA and poly(dG)-poly(dC) DNA corresponding to sequentially changed Asp values were collected. For simplicity, three images that correspond to stages (1), (2), and (3) are shown in parts b-d of Figure 2. Langmuir 2010, 26(10), 7523–7528

In this study, an improved equation for the height calculation was developed, which can replace the approximated equation used in our previous studies.13,21 In comparison with that used previously, in the newly developed equation the influence of the change of oscillation amplitude with the change of Asp value (unit: volts) on the height measurement has been incorporated, allowing a more accurate height measurement of the molecule to be obtained. Under VSPFM the height of a given location on the G4-DNA segment in the jth step compression can be calculated by Dj ¼ Dj-app þ Dj-s - cðAspðjÞ - AspðbottomÞ Þ

ð1Þ

where Dj-app denotes the apparent height of G4-DNA, Dj-s is the displacement of the rest position of the cantilever vertically when the Asp value changes from Asp(j) to Asp(bottom) (the last step in the compression in which the tip touches the substrate) and can be obtained from the zero-scale scan at a DNA-free area of the substrate (Figure 2e) and Z-profile analysis (Figure 2f),21 and c (nm/V) is the conversion coefficient of the Asp value (in the region of Asp(0) to Asp(bottom)) from volts to nanometers in TM-AFM, which can be obtained from the force calibration plot (Figure 3). Asp(0) is the Asp value in the 0th step compression where the tip just starts touching the top of the target location on G4-DNA and is also termed Asp(top) in Figure 2a. The last term (c(Asp(j) Asp(bottom))) in the equation is used to calculate the change of oscillation amplitude with a decrease of Asp value. The combination of the last two terms (Dj-s - c(Asp(j) - Asp(bottom))) in the equation denotes the tip-to-substrate distance at Asp(j). As demonstrated by the Z-profile analysis (Figure 2f), the displacement of the tip can be precisely controlled through changing Asp values with no evidence of drift in the Z-direction being observed during the process of compressing the molecules, which is crucial to the accurate measurement of the molecules’ heights. The height of the target location on the poly(dG)-poly(dC) DNA can be calculated following the same methodology as for calculating the height of G4-DNA, but the corresponding parameters in the compression of the poly(dG)-poly(dC) segment need to be used. The average height of G4-DNA measured by VSPFM was ∼1.8 nm, which is ∼30% higher than the value measured by DOI: 10.1021/la904329q

7525

Article

Wang et al.

Figure 2. (a) Schematic representation of using VSPFM to compress G4-DNA (yellow oval) and poly(dG)-poly(dC) DNA (blue oval) segments at sequentially changed Asp values (Asp(top), Asp(j), and Asp(bottom)). D0-app, Dj-app, and Dbottom-app are apparent heights of a certain point on the G4-DNA segment at corresponding Asp values. D0 0-app and D0 bottom-app are apparent heights of a certain location on the poly(dG)-poly(dC) DNA segment at Asp(j) and Asp(bottom), respectively. For simplicity, the green lines are used to denote the trace profile of the tip during the scanning process, in which the tip should keep oscillating at a certain Asp while moving along the trace profile. It should be noted that the objects illustrated in the schematics are not to scale. (b-d) VSPFM height images (scan size: 160  80 nm2) taken during the process of compressing G4-DNA (the brighter molecule) and poly(dG)-poly(dC) DNA molecules, which are corresponding to stages (1)-(3) in (a). The values of Asp are (b) 0.81, (c) 0.79, and (d) 0.74 V. (e) Zero-scale scan image21 at a DNA-free area of the substrate. Asp varies uniformly from 0.81 to 0.74 V from the top to the bottom; i.e., the Asp decreases 0.01 V in each step. The displacements of the rest position of the cantilever relative to the substrate are shown at the corresponding steps. (f) Z-profile along the black line in (e), indicating the rest position of the tip moves downward 2.56 nm before touching the substrate.

Figure 3. Typical force calibration plot in TM-AFM. The red and black curves denote the retracting and extending amplitude-Z position curves, respectively. The Asp values used in our experiment usually were in the region between the green line (Asp value: 0.81 V) and the blue line (Asp value: 0.71 V). The curves between the green and blue lines are approximately linear, and the inverse of the slope of the curves in this region is termed the conversion coefficient c, ∼10 nm/V.

TM-AFM, while the height of poly(dG)-poly(dC) DNA measured by VSPFM was ∼1.3 nm, which is ∼85% higher than the value measured by TM-AFM. These results confirm that soft molecules can be easily deformed by the tip-sample interactions in the imaging process in TM-AFM under ambient conditions. However, the heights measured by VSPFM are still less than their 7526 DOI: 10.1021/la904329q

nominal diameters. These discrepancies may be partly due to the deformations induced by the adsorption between the molecule and the substrate as well as the inevitable slight tip-sample pressure when the tip initially touches the top of the molecules in the VSPFM operation.21 In addition, other factors such as the roughness of the substrate and possible dehydration of the molecules may also affect the measured heights.21 In Figure 4b, several typical height-force curves corresponding to different sites (indicated by the arrows in Figure 4a) along G4-DNA and poly(dG)-poly(dC) DNA are shown. The positions chosen for the mechanical analysis are those with a height around the average height of the DNA molecules in TM-AFM (0.6-0.9 nm for poly(dG)-poly(dC) DNA and 1.2-1.5 nm for G4-DNA). Singular points such as “kink” points (notably higher locations) were avoided because that they might be caused by intrachain interactions of the DNA molecules and do not represent the natural structure of the DNA molecules.39 These curves show a nonlinear compression behavior with applied loads. Clearly, when the force is between ∼0.04 and ∼0.12 nN, the deformation of these molecules changes quickly, whereas when the force is higher than ∼0.12 nN, the deformation changes much more slowly. This is indicative of the extreme softness at the initial compression and also implies that there is a transition in the deformation mechanism. The force constants of the molecules at lower loading forces (F < ∼0.12 nN), estimated by ΔF/Δh, are (39) Li, J. W.; Bai, C. L.; Wang, C.; Zhu, C. F.; Lin, Z.; Li, Q.; Cao, E. H. Nucleic Acids Res. 1998, 26, 4785–4786.

Langmuir 2010, 26(10), 7523–7528

Wang et al.

Article

DNA molecules is highly reversible, as we demonstrated in our previous work.13 The deformation properties of poly(dG)poly(dC) DNA are consistent with λ-phage DNA.13 Through force-strain curves41 in Figure 4c, the deforming properties of G4-DNA and poly(dG)-poly(dC) DNA are more clearly depicted. When the force is larger than ∼0.12 nN, the strain of poly(dG)-poly(dC) DNA is obviously higher than G4-DNA under the same loading force. For quantitative comparison of the rigidity of G4-DNA and poly(dG)-poly(dC) DNA, the effective radial elastic moduli of these two molecules were estimated by E ¼ ðdF=dhÞðh=SÞ

ð2Þ

Here, h and S are the molecular height and the contact area between the tip and the molecule at a given compressing force, F.41 Based on Hertz’s theory of normal contact of two elastic solids (a spherical tip and a cylindrical tube), the S could be calculated by S ¼ 2πΔhRtip ðRm =ðRm þRtip ÞÞ1=2

Figure 4. (a) Three-dimensional image of G4-DNA and poly(dG)-poly(dC) DNA segments in TM-AFM (scan size: 200  200 nm2, Z scale: 5 nm), in which the green arrows marked the locations that were analyzed. (b) Height-force curves and (c) force-strain curves calculated for G4-DNA and poly(dG)-poly(dC) DNA segments at different locations as marked in (a). The legends A, B, C, D, E, F, G, H, and I correspond to the marked locations in (a).

∼0.2-0.3 N/m, where ΔF and Δh denote the increment of the force loading and the corresponding decreased height of the molecules, respectively. It is likely that “weak” forces, such as hydrogen bonding, electrostatic, and van der Waals interactions, are involved in this deformation regime.40 The “weak” forces maintain biological structure, which renders biological molecules “soft” at room temperature.40 The slower deformation of the molecules at higher loading forces (F > ∼0.12 nN) is probably related to squeezing of the backbones of the molecules, which leads to reduced space between the backbones and thus stronger repulsive forces. In our experiment, the radial deformation of the (40) Zaccai, G. Science 2000, 288, 1604–1607.

Langmuir 2010, 26(10), 7523–7528

ð3Þ

where Rm and Rtip are the radius of the molecule and the tip, respectively (Rm is a half of the height of the molecule under VSPFM).42 Based on previous studies, the influence of the substrate on the elastic modulus can be neglected in the regime we are interested in.13,41 From the curves in Figure 4c, the values of the radial compressive elastic modulus for the two DNA derivatives were estimated to be ∼5-10 MPa when the external force is between ∼0.04 and ∼0.12 nN and increased to ∼10-20 MPa for poly(dG)-poly(dC) DNA and ∼20-40 MPa for G4DNA when the force is larger than ∼0.12 nN. Although there are some deviations of the absolute modulus values between different experiments, the compression performance of the two molecules as shown in Figure 4 are highly reproducible. G4-DNA is a tetrahelical structure composed of sets of fourbase units, rather than the usual double-helical DNA molecules composed of base pairs.43 The planar G-tetras resemble macrocycles, which stack together to form a column structure (G4DNA) with a central cavity.44 STM studies have shown that the distance between the G-quartet stacks of G4-DNA is ∼0.35 nm,7 whereas the distance between the base pairs poly(dG)-poly(dC) DNA is ∼0.38 nm.8 Considering their similar nominal diameters, one can understand intuitively that G4-DNA should possess less spaces between the backbones, in comparison with poly(dG)-poly(dC) DNA. It is conceivable that the different architectures of these two kinds of molecules contribute significantly to their deforming properties. Our results are consistent with previous studies in which it was proposed that G4-DNA was stiffer than dsDNA molecules because G4-DNA appeared much higher than dsDNA molecules in the AFM image, in spite of their similar nominal diameters.4 We anticipate that computer simulations would be helpful to gain a deeper understanding of the underlying mechanism of deformation.

Conclusion In summary, VSPFM has been used to evaluate the radial compression elasticity of G4-DNA and poly(dG)-poly(dC) (41) Yu, M. F.; Kowalewski, T.; Ruoff, R. S. Phys. Rev. Lett. 2000, 85, 1456– 1459. (42) Shen, W. D.; Jiang, B. Phys. Rev. Lett. 2000, 84, 3634–3636. (43) Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668–698. (44) Phillips, K.; Dauter, Z.; Murchie, I. H. A.; Lilley, M. J. D.; Luisi, B. J. Mol. Biol. 1997, 273, 171–182.

DOI: 10.1021/la904329q

7527

Article

DNA. The radial compressive elastic modulus shows an interesting nonlinear increase with the applied loading force due to a transition in the deformation mechanism of the DNA molecules. When the force is larger than ∼0.12 nN, the difference in the elastic moduli between G4-DNA and poly(dG)-poly(dC) DNA is distinguishable and G4-DNA is stiffer than poly(dG)-poly(dC) DNA. We report here the first success of distinguishing the mechanical responses of two similar molecules subjected to a load lower than 0.4 nN. These results are instructive for single DNA molecule nanomanipulation such as cutting, kneading, and picking up a DNA segment with an AFM tip in air.45,46 By combining the AFM-based nanomanipulation with single-molecules PCR, we have successfully sequenced single DNA molecules.45,46 These results are also valuable in interpreting deformation-related electronic properties of these molecules and in providing information (45) An, H. J.; Huang, J. H.; L€u, M.; Li, X. L.; L€u, J. H.; Li, H. K.; Zhang, Y.; Li, M. Q.; Hu, J. Nanotechnology 2007, 18, 225101. (46) L€u, J. H.; Li, H. K.; An, H. J.; Wang, G. H.; Wang, Y.; Li, M. Q.; Zhang, Y.; Hu, J. J. Am. Chem. Soc. 2004, 126, 1136–1137.

7528 DOI: 10.1021/la904329q

Wang et al.

on their deformability that is necessary in designing DNAbased molecular devices as well as in developing a deeper understanding of the biological functions of poly(dG)poly(dC) and G-quadruplex-forming sequences. It is postulated that, coupled with an ultrasharp conductive tip (e.g., a conductive carbon nanotube tip),6 VSPFM could be used to provide even higher definition information on the deformation behavior of individual functional molecules, which would render VSPFM a valuable technique in the field of nanotechnology. Acknowledgment. We thank Dr. Nobuo Maeda and Mr. Adam Brotchie for helpful comments on this manuscript. We also gratefully acknowledge the anonymous reviewers for the insightful comments. This work was funded by the National Natural Science Foundation of China (30800255 and 10604034) and the Science and Technology Ministry of China (2007CB936000 and 2006CB932505). X. Zhang is the recipient of Australian Postdoctoral Fellowship (DP0880152).

Langmuir 2010, 26(10), 7523–7528