4224-4228

Nucleic Acids Research, 1994, Vol. 22, No. 20

1994 Oxford University Press

Stretched DNA structures observed with atomic force microscopy

Thomas Thundat*, David P.Allison and Robert J.Warmack Health Sciences Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6123, USA

Received June 13, 1994; Revised and Accepted September 6, 1994

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ABSTRACT Double-stranded DNA molecules are occasionally found that appear to be straightened and stretched in atomic force microscope (AFM) images. Usually pBS + plasmid and lambda DNA show relaxed structures with bends and kinks along the strands and have measured contour lengths consistent to about 5 - 7 % ; they also appear not to cross over each other, except in very high concentrations. The anomalous molecules observed here, compared with the majority of molecules in the preparation, show contour lengths increased by as much as 80% and have measured heights of about half that of normal relaxed DNA. Some molecules also appear to be in transition between stretched and relaxed forms. These observations are consistent with an uncoiling of the DNA helix without breakage of the covalent bonds in the deoxyribose-phosphate backbone. INTRODUCTION AFM images of DNA mounted on a mica surface generally show a relaxed polymer with broadened width and reduced height that curves, bends, and kinks as it lies over the surface. In early reports of AFM imaging of DNA on mica, the DNA strands were often tangled in aggregates (1), but with the use of magnesium during DNA deposition relaxed, isolated strands are typical (15). Critical point drying appears to further improve the mounting and imaging of DNA (6). Curiously, DNA strands mounted this way very rarely cross each other unless the concentration is very high. The lengths of DNA molecules measured from AFM images are consistently within 57% of the calculated lengths (4,7-9). The observed widths of DNA molecules in AFM images are larger than the known molecular width of DNA due to the well known broadening caused by the finite probe tip radius (1-14). However, the reduced height of DNA molecules observed with the atomic force microscope remains a puzzle. All reported AFM images show measured DNA heights much smaller than the expected value of 2 nm (1 - 1 4 ) . Bustamante et al. (4) attribute reduced height to DNA deformation under the AFM tip while Vesenka et al. (13) attribute the decreased heights of DNA to the presence of buffer salts. Large adhesion forces between the
*To whom correspondence should be addressed

probe tip and DNA probably also tend to reduce the height (14,15). Lateral forces due to increased relative humidity has as well been found to have a role in the observed height differences (16). In general, however, the heights of the DNA molecules in individual images do not vary appreciably from one to another. In this paper we present AFM images of unusual DNA molecules that appear to be stretched greatly beyond normal and also have reduced heights compared with the surrounding molecules. The stretched structures we find are more prevalent for long DNA molecules such as lambda phage DNA, but could also be infrequently found in circular /?BS+ plasmid preparations. We attribute this to mechanical forces introduced during sample preparation. These unusual structures are found when the samples are prepared by either air drying or critical point drying.

MATERIALS AND METHODS Sample preparation

DNA used in this study was /?BS + (3204 bp, from Stratagene, LaJolla, CA) and lambda DNA (47 kbp, Sigma, St Louis, MO) diluted into 0.01 M ammonium acetate buffer (Fisher Analytical reagent grade) at pH 7.2. The mounting substrates were freshly cleaved muscovite mica (New York Mica Co., New York, NY). In some preparations, pBS + DNA had been linearized by treatment with Smal and in other preparations the DNA was partially digested with EcoKL restriction enzyme. Samples were prepared for AFM imaging by placing a 25 fi\ drop ofO. 15 to 0.45 jtg/ml DNA solution containing 5 - 1 0 mM magnesium acetate onto freshly cleaved mica for 10 minutes. The samples were then rinsed for 10 seconds in a jet of distilled water directed onto the surface with a squeeze bottle, plunged into ethanol-water mixture (1:1) five times, followed by plunging five times each in three changes of 100% ethanol. Some of the samples were blown dry with dry nitrogen while others were critical point dried (Autosamdri-810 critical point dryer, Tousimis Research Corp., Rockville, MD). This latter procedure takes approximately 30 minutes and replaces ethanol with liquid CO2 that is passed through the liquid to gas phase at the critical point. The dried samples were then kept in a desiccator until use.

Nucleic Acids Research, 1994, Vol. 22, No. 20 4225 Atomic force microscopy AFM images were collected using commercially available contact and tapping mode instruments (Digital Instruments, Inc., Santa Barbara, CA). The contact scanning tips used were silicon nitride (Si3N4) cantilevers, 200 /un long with a nominal spring constant of 0.12 N/m. The tapping mode tips used were rectangular silicon cantilevers with a spring constant of 30 N/m. Contact AFM images were obtained in a nitrogen atmosphere in a constant force mode (310 nN net repulsive force on the cantilever) and are presented here as raw data except for flattening. The tapping mode AFM was operated at a scan rate of 1.5 Hz in a dry helium atmosphere for improved resonant behavior (17). Typical resonant frequencies were about 310 kHz with a quality factor around 600. The dimensional measurements of DNA were made by taking line sections to obtain the height information or by following the contour of molecule with point-to-point cursors in the Nanoscope software to obtain lengths. The contour lengths of plasmids were also obtained using digital analysis software (NIH Image, National Institute of Health, Bethesda, MD), which provided better statistical data. The AFM scanner calibration was verified to 4% in x andj>, and to 10% in z using a calibration grating supplied by the manufacturer. RESULTS In general, tapping mode AFM images of DNA molecules adsorbed on mica show bends and kinks that appear to be randomly distributed along the strands. Occasionally, however, straight molecules or straight sections of DNA molecules are seen. Figure 1 shows images taken of lambda DNA fragments prepared by criticaj point drying that show both relaxed and straightened DNA. These latter molecules also show other noticeable features: decreased heights and increased lengths. The same qualitative differences were observed in both tapping mode and contact mode AFM. Measured heights of DNA strands From our experience in mounting hundreds of DNA samples, the measured height of relaxed DNA remains constant for a given sample and scanning conditions. Except for the straightened segments, all of the molecules in images taken on the same sample show the same heights. For example, the observed height of the relaxed molecules in Figure 1 is 0.79 0.05 nm, well below 2 nm but consistent with the reported heights of DNA between 0.1-1.5 nm. Most of these fragments shown in Figure la show relaxed strands with bends and kinks while one fragment shows both straightened and relaxed regions. Figure lb shows the continuation of the straightened fragment shown in Figure la. This particular fragment of DNA was chosen because it shows straightened sections both parallel and perpendicular to the scan

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Figure 1. A series of tapping mode AFM images of lambda DNA fragments adsorbed on mica prepared by critical point mounting where both straightened and relaxed regions coexist. Note that in (a) and (b) the straight regions are at an angle roughly parallel and perpendicular to the scan direction. Figure (c) is a higher magnification image of the lower right corner of (b) where the measured heights of straight regions are -50% lower than the relaxed regions.

4226 Nucleic Acids Research, 1994, Vol. 22, No. 20 directions. This demonstrates the typical finding that neither the presence of anomalous molecules nor the height anomalies observed were related to the scan direction. The average height of the stretched fragment is 0.45 0.04 nm or about half that of the relaxed DNA. Figure lc is a high magnification image of Figure lb showing the height difference and point where relaxed and straightened DNA cross over each other. Measured widths of DNA molecules The apparent width of DNA was larger than the 2 nm intrinsic width of DNA due to radius of the contacting tip and large adhesion forces. The widths of relaxed and straightened DNA molecules in Figure 3b are 21.9 3.7 nm and 18.7 1.9 nm respectively. Though the widths of DNA molecules shown in this paper are rather large, we were able to get DNA widths as small as 9 nm at times. Measured DNA lengths We measured the lengths of both the anomalous as well as the relaxed molecules. Figure 2 shows the contour lengths resulting from the digital analysis of more than two hundred images of both circular and linearized pBS+ plasmids. Although a distribution of sizes is observed, the peak is at 972 nm for the circular form and 957 nm for the linear form. This is very close to 0.34 nm/base pair expected for B-form DNA in solution (18). The standard deviation is 5.3% and 5.6% respectively (Figure 2). Figure 3 shows typical images of the anomalously straightened circular and linear pBS+ DNA. The length of the relaxed DNA in this preparation was consistent with the above statistical measurements. However, the lengths of stretched circular DNA molecules were 2030% larger (Figure 3a), and the linearized DNA was up to 80% longer than the relaxed variety (Figure 3b). We found many molecules that appeared to be stretched along portions of their lengths and were significantly longer than relaxed DNA, but shorter than the maximum observed. We found a greater prevalence of the anomalous DNA in longer molecules such as lambda DNA than in shorter linearized plasmids and the least amount in the closed circular plasmid form. The lengths of straightened lambda fragments were not measured since many fragments are produced in a partial coRI digestion and there would be no way of knowing the relative lengths of the straightened and the relaxed fragments. Whole lambda DNA molecules were also found to be straightened in sections rather than over their entirety. Because of the surface coverage and confusion at crossover points it was not possible to measure and compare partially straightened molecules to relaxed lambda DNA.

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60

S 40
3
V

pBS+ (circular)

30 20 10
^jl

200 60
< u 40 3

400 600 800 1000 1200

pBS + (linear)

cr

2 20

--'-1
200 400 600 BOO 1000 1200

Length (nm)

Figure 2. Frequency histograms from AFM length measurements of circular form pBS + (a) and linearized pBS + (b). The average lengths and standard deviations 973 51 nm for (a) and 958 53 nmfor(b).

Figure 3. Tapping mode AFM images of pBS+ plasmid DNA prepared by critical point drying. The lengths of elongated circular molecules (a) were 30"c greater than the lengths of relaxed circular DNA. In pBS* DNA linearized with Sma\ (b) the elongated strand was 78% longer than the relaxed linear DNA molecules. Note that relaxed unstretched molecules of DNA appear to cross over stretched strandi.

Nucleic Acids Research, 1994, Vol. 22, No. 20 4227 Repulsion between DNA strands An interesting feature of our preparations with moderate surface coverage is that the relaxed strands very rarely cross each other. Figure 4a shows AFM images of lambda DNA fragments prepared by critical point drying where a few of the fragments appear to be stretched. The relaxed DNA strands appear to avoid crossing each other but the anomalous strands intersect the relaxed molecules in an unfettered fashion. Figure 4b shows a typical AFM image of circular pBS+ where none of the DNA strands cross each other. The anomalous strands do not appear to follow this rule; they cross the relaxed strands freely. DISCUSSION Increases in DNA lengths of up to 80% may appear to be unrealistic and well above elastic limits. Recent elastic studies of DNA by Smith et al. (19) report no significant increases in length of DNA in solution even when individual molecules were pulled by forces up to 10" n N. DNA is tightly coiled due to coulombic, van der Waals, and molecular orbital forces. However, the deoxyribose-phosphate backbones are strongly joined by covalent bonds. It is conceivable that with sufficient external forces DNA can be stretched or uncoiled without breaking the backbones. If DNA is modeled as a 2 nm wide ladder with a helical repeat of 3.4 nm, an increase in length of over a factor of two is possible by uncoiling the helix without breaking the phosphate bonds (see Figure 5). It may also be possible to stretch the DNA molecule without uncoiling. In this case the diameter of the helix must decrease. This would amount to 12% for a 30% stretch and 43% for an 80% stretch. Large changes in base pair distances are clearly unreasonable, so that some uncoiling must occur for the longer elongations. Linear molecules would have the freedom rotating to allow uncoiling and were found to be more elongated. Circular molecules had straightened sections but were less elongated. We speculate that some stretching and uncoiling may occur in different proportions for various conditions. Deformation may take place during rinsing in a jet of water where hydrodynamic forces pull on molecules that are not well anchored to the substrate at all points along the molecular length. Though Shaiu et al. observed the aligning of DNA molecules during rinsing, they do not report any increase in DNA length (20). In our images of anomalous molecules, the straightened strands were aligned generally in the same direction. Unfortunately, sample alignment was not preserved during the rinsing steps, so orientation with rinse direction cannot be verified. If they were elongated, one would think that possibly the stored mechanical energy could be released by cutting the strand into two pieces (this was achieved by increasing the contact force and scanning back-and-forth across the strand). However, such dissection of these molecules did not make them spring back, indicating that they must be either attached continuously to the surface or tacked down at the point of dissection. Continuous attachment may also be indicated by the fact that we have observed some lambda fragments in the shape of a parabola or a semicircle.

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[ 2.0j

2.0

JT

Figure 4. AFM image of (a) fragments of lambda DNA and (b) pBS+showing possible electrostatic repulsion between the strands. The relaxed DNA strands do not interact and remain separate from one another. In the lambda DNA preparation (a) some of the DNA strands appear to be straightened. Note that unstretched relaxed DNA fragments freely cross the anomalous DNA.

Figure 5. A schematic showing the relationship between the length of normal B-form DNA, 3.4 nm helical repeat (10 bp), and the possible length of the completely uncoiled molecule without breaking the phosphate linkages. Assuming a helical diameter of 2.0 nm, the maximum geometric extension would be 7.1/3.4 or 2.1 times the normal length.

4228 Nucleic Acids Research, 1994, Vol. 22, No. 20 The widths of DNA observed by AFM may be modified by adsorbed salt. When imaging was done at higher humidity ( > 50%), apparent widths of relaxed DNA were found to increase as much as 100% possibly due to the hygroscopic nature of buffer salt around the DNA strands (16). However, the anomalous DNA did not show any appreciable increase in width at higher relative humidity. Also, if die stretched DNA has a reduced layer of salt, its linear charge density may be different. This charge should also be affected by the lengthening process itself. Since AFM tip deflection is due to a number of forces acting on the tip including electrostatic interaction, changes in the dipole interaction could contribute to the observed heights, especially for DNA because of its small diameter and high linear charge density. The decrease in height of DNA was found to depend on the extent of lengthening, with molecules whose contour lengths showed only slight lengthening being less affected than molecules that were highly lengthened. For example, the molecules that are elongated only 30% do not show any appreciable change in measured heights. However, molecules that are stretched 80% show a height difference of up to 50% between straightened and relaxed regions. A gradient in height can also be observed in the areas of transition in the same molecules (Figure lb). The fact that relaxed DNA strands very rarely cross indicates that some repulsive force between the strands is operating as the DNA is being deposited. Repulsive forces acting after the surface has been covered would leave some strands crossing and in an unstable equilibrium which we do not see. Since the repulsion, as evidenced by the proximity of one DNA strand to another (Figure 4), appears to act over long range (up to - 5 0 nm), the interaction force is probably electrostatic in nature. The anomalous strands do not appear to be affected by the relaxed DNA lying nearby. We conclude that there is no significant interaction between them, at least when the DNA is being deposited. If both types were laid down simultaneously, this would indicate that the anomalous DNA is electrostatically neutral. We speculate that the two strands are electrostatically different, and that the anomalous DNA has lost its net charge. REFERENCES
1. Thundat, T., Allison, D.P., Warmack, R.J., Brown, G.M., Jacobson, K.B., Schrick, J.J., and Ferrell, T.L., (1992) Scanning Microsc. 6, 911-918. 2. Vesenka, J., Guthold, M., Tang, C.L., Keller, D., Delaine, E., Bustamante, C , (1992) Ultramicroscopy 42-44, 1243-1249. 3. Henderson, E., (1992) Nucleic Acids Res. 21, 445-447. 4. Bustamante, C , Vesenka, J., Tang, C.L., Rees, W., Guthold,M, Keller, R., (1992) Biochem. 31, 22-26. 5. Hansma, H.G., Vesenka, J., Siegerist, C , Kelderman, G., Morrett, H., Sinsheimer, R.L., Elings, V., Bustamante, C , and Hansma, P.K., (1992) Science 256, 1180-1184. 6. Thundat, T., Allison, D.P., Warmack, R.J., and Jacobson, K.B., (1994) Scanning Microsc. 8, 2330. 7. Thundat.T., Allison, D.P., Warmack, R.J., Doktyz, M.J., Jacobson, K.B., and Brown, G.M., (1993) J. Vac. Sci. Technol. A l l , 824-828. 8. Hansma, H.G., Bezannilla, M., Zenhausem, F., Adrian, M., and Sinsheimer, R.L., (1993) Nucleic Acids Res. 21, 505-512. 9. Lyubchenko, Y.L, Jacobs, B.L., and Lindsay, S.M., (1992) Nucleic Acids Res. 20, 3983-3986. 10. Thundat, T., Allison, D.P., Warmack, R.J., and Ferrell, T.L., (1992) Ultramicroscopy 42-44, 1101 -1106. 11. Allen, M.J., Hud, N.V., Balooch, M., Tench, R.J., Siekhaus, W.J., and Ballhorn, R., (1992) Ultramicroscopy 42-44, 1095-1100. 12. Zenhausern, F., Adrian, M., ten Hieggeler-Bordier, B., Emch, R., Jobin, M., Taborelli, M., and Descouts, P., (1992) J. Struct. Biol. 108, 6 9 - 7 3 . 13. Vesenka, J., Manne, S., Yang, G., Bustamante, C , and Henderson, E., (1993) Scanning Microsc. 7, 781-788. 14. Lyubchenko, Y.L., Oden, P.I., Lampner, D., Lindsay, S.M., and Dunker, K.A., (1993) Nucleic Acids Res. 21, 1117-1123. 15. Yang, J. and Shao, Z., (1993) Ultramicroscopy. 50, 157-170. 16. Thundat, T., Warmack, R.J., Allison, D.P., Bottomley, L.A., Lourenco, A.J., and Ferrell, T.L., (1992) J. Vac. Sci. Technol. A10, 630-635. 17. Chen, G.Y., Warmack, R.J., Thundat, T., Allison, D.P., Huang, A., (1994) Rev. Sci. Instrum. 65, 2532-2537. 18. Watson, J.D., Hopkins, N.H., Roberts, J.W., Steitz, J.A., Weiner, A.M., (1988) Molecular Biology of the Gene Fourth edition, p249. Benjamin/Cummings Publishing Co., Inc. Menlo Park, CA. 19. Smith, S.B., Finzi, L., and Bustamante, C , (1992) Science 258, 1122-1126. 20. Shaiu, W.L., Larson. D.D., Vesenka, J., and Henderson, E., (1993) Nucleic Acid Res. 21, 99-103.

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CONCLUSION Unusual strands frequently appear in AFM images of plasmid and lambda DNA that have been adsorbed onto mica, vigorously rinsed, and dried. These strands differ from the usual relaxed strands in several ways. Their lengths are up to about 80% longer and their heights are about 50% less than relaxed strands. These observations are consistent with stretching and uncoiling of the DNA helix possibly induced by rinsing followed by pinning to the mica surface. Whereas the relaxed DNA strands appear to have a linear charge density that prevents them from crossing each other, the anomalous strands cross freely.

ACKNOWLEDGEMENTS We would like to thank M.C.Rorvik, K.B.Jacobson, J. Vesenka and B.Hingerty for many helpful comments, and D.Englehart for measuring apparent DNA lengths. This research was sponsored by the Office of Health and Environmental Research, US Department of Energy under contract DE-AC05-84OR21400 with Martin Marietta Energy- Systems, Inc.