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4224 4228  Nucleic Acids Research, 1994, Vol. 22, No. 20 © 1994 O xford University Press

Stretched DNA structures observed with atomic forcemicroscopy

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

ABSTRACT

Double-stranded DNA molecules are occasionally

found that appear to be straightened and stretched inatomic force microscope (AFM) images. Usually pBS +

plasmid and lambda DNA show relaxed structures withbends and kinks along the strands and have measuredcontour lengths consistent to about 5 - 7 ; they alsoappear not to cross over each other, except in very highconcentrations. The anomalous molecules observedhere, compared with the majority of molecules in thepreparation, show contour lengths increased by asmuch as 80 and have measured heights of about halfthat of normal relaxed DNA. Some molecules alsoappear to be in transition between stretched andrelaxed forms. These observations are consistent withan uncoiling of the DNA helix without breakage of the

covalent bonds in the d eoxyribose-phosph atebackbone.

INTRODUCTION

AFM images of DNA mounted on a mica surface generally showa relaxed polymer with broadened width and reduced height thatcurves, bends, and kinks as it lies over the surface. In earlyreports of AFM imaging of DNA on mica, the DNA strands wereoften tangled in aggregates (1), but with the use of magnesiumduring DNA deposition relaxed, isolated strands are typical(1—5).  Critical point drying appears to further improve themounting and imaging of DNA (6). Curiously, DNA strands

mounted this way very rarely cross each other unless theconcentration is very high.

The lengths of DNA molecules measured from AFM imagesare consistently within 5—7% of the calculated lengths (4 ,7 -9 ).The observed widths of DNA molecules in AFM images arelarger than the known molecular width of DNA due to the wellknown broadening caused by the finite probe tip radius (1-14).However, the reduced height of DNA molecules observed withthe atomic force microscope remains a puzzle. All reported A FMimages show measured DNA heights much smaller than theexpected value of 2 nm   1 -1 4 ). Bustamante et al.  (4) attributereduced height to DNA deformation under the AFM tip whileVesenka  et al.  (13) attribute the decreased heights of DNA tothe presence of buffer salts. Large adhesion forces between the

probe tip and DNA probably also tend to reduce the height

(14,15). Lateral forces due to increased relative humidity hasas well been found to have a role in the observed heightdifferences (16). In general, however, the heights of the DNAmolecules in individual images do not vary appreciably from oneto another.

In this paper we present AFM images of unusual DNAmolecules that appear to be stretched greatly beyond normal andalso have reduced heights compared with the surroundingmolecules. The stretched structures we find are more prevalentfor long DNA molecules such as lambda phage DNA, but couldalso be infrequently found in circular /?BS +  plasmidpreparations. We attribute this to mechanical forces introducedduring sample preparation. These unusual structures are foundwhen the samples are prepared by either air drying or critical

point drying.

M A T E R IA L S A N D M E T H O D S

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 Analyticalreagent grade) at pH 7.2. The mounting substrates were freshlycleaved muscovite mica (New York Mica Co., New York, NY).In some preparations, pBS +  DNA had been linearized bytreatment 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 mMmagnesium acetate onto freshly cleaved mica for 10 minutes.The samples were then rinsed for 10 seconds in a jet of distilledwater directed onto the surface with a squeeze bottle, plungedinto ethanol-water mixture (1:1) five times, followed byplunging five times each in three changes of 100% ethanol. Someof the samples were blown dry with dry nitrogen while otherswere critical point dried (Autosamdri-810 critical point dryer,Tousimis Research Corp., Rockville, MD). This latter proceduretakes approximately 30 minutes and replaces ethanol with liquidCO 2 that is passed through the liquid to gas phase at the criticalpoint. The dried samples w ere then kept in a desiccator until use.

*To whom correspondence should be addressed

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Nucleic Acids Research, 1994, Vol. 22, No. 20  4225

Atomic force microscopy

AFM images were collected using commercially available contactand tapping mode instruments (Digital Instruments, Inc., SantaBarbara, C A). The contact scanning tips used were silicon nitride(Si3N4) cantilevers, 200 /un long with a nominal spring constantof 0.12 N/m . The tapping mode tips used w ere rectangular siliconcantilevers with a spring constant of 30 N/m. Contact AFMimages were obtained in a nitrogen atmosphere in a constant forcemode (3—10 nN net repulsive force on the cantilever) and arepresented here as raw data except for flattening. The tappingmode AFM was operated at a scan rate of 1.5 Hz in a dry heliumatmosphere for improved resonant behavior (17). Typicalresonant frequencies were about 310 kHz with a quality factoraround 600.

The dimensional measurements of DN A were made by takingline sections to obtain the height information or by following thecontour of molecule with point-to-point cursors in the N anoscopesoftware to obtain lengths. The contour lengths of plasmids were

also obtained using digital analysis software (NIH Image, NationalInstitute of Health, Bethesda, MD), which provided betterstatistical data. The AFM scanner calibration was verified to± 4 %  in x  andj>, and to ±10% in z using a calibration gratingsupplied by the manufacturer.

RESULTS

In general, tapping mode AFM images of DNA moleculesadsorbed on mica show bends and kinks that appear to berandomly distributed along the strands. Occasionally, however,straight molecules or straight sections of DNA molecules areseen. Figure 1 shows images taken of lambda DN A fragmentsprepared by criticaj point drying that show both relaxed and

straightened DNA. These latter molecules also show othernoticeable features: decreased heights and increased lengths. Thesame qualitative differences were observed in both tapping m odeand contact mode AFM.

Measured heights of DNA strands

From our experience in mounting hundreds of DNA samples,the measured height of relaxed D NA remains constant for a givensample and scanning conditions. Except for the straightenedsegments, all of the molecules in images taken on the same sampleshow the same heights. For example, the observed height of therelaxed molecules in Figure  1 is 0.79 ± 0.05 nm , well below2 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 bothstraightened and relaxed regions. Figure lb shows thecontinuation of the straightened fragment shown in Figure la.This particular fragment of DNA was chosen because it showsstraightened sections both parallel and perpendicular to the scan

Figure 1. A  series of tapping mode AFM images of lambda D NA fragmentsadsorbed on mica prepared by critical point mounting where both straightenedand 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) isa higher m agnification image of the lower right corner of  b) where the measuredheights of straight regions are -50%  lower than the relaxed regions.

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4226  Nucleic Acids Research, 1994, Vol. 22, No. 2

directions. This demonstrates the typical finding that neither thepresence  of  anomalous molecules  nor the height anomaliesobserved were related to the scan direction. The average heightof the stretched fragment is 0.45  ±  0.04 nm or about half thatof the relaxed DNA. Figure  lc is a high m agnification image

of Figure  lb showing th e height difference and point w hererelaxed and straightened DNA cross over each other.

Measured widths of DNA molecules

The apparent width of DNA was larger than the 2 nm intrinsicwidth  of  DNA due  to radius  of the contacting tip and largeadhesion forces. The widths of relaxed and straightened DNAmolecules in Figure 3b are 21.9  ±  3.7 nm and 18.7  ±  1.9 nmrespectively. Though the widths of DNA molecules shown inthis paper are rather large, we were able to get DNA widths assmall as 9 nm at times.

Measured DNA lengths

We measured the lengths of both the anomalous as well as therelaxed molecules. F igure 2 shows the contour lengths resultingfrom the digital analysis of more than two hundred images ofboth circular  and linearized  pBS

+  plasmids. Although  a

distribution of sizes is observed, the peak is at 972 nm for thecircular form and 957 nm for the linear form. This is very closeto 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 straightenedcircular and linear pBS+  DNA . The length of the relaxed DNAin this preparation was consistent with  the  above statisticalmeasuremen ts. Ho wever, the lengths of stretched circular DNAmolecules were 20—30% larger (Figure 3 a), and the linearizedDNA was up to 80% longer than the relaxed variety (Figure 3b).We found many molecules that appeared to be stretched alongportions o f their lengths and were significantly longer than relaxedDNA, but shorter than the maximum observed.

60

S  40

3  3

V  2

^  1

60

<u  43cr2  2

pBS+

  circular)

+ +^ j l

200  4 6 8 1000  12

pBS +

(linear)

-- -1200  4 6 BOO 1000  12

Length (nm)

Figure 2. Frequency histograms from AFM length measurements of circular formp B S +  (a) and linearized pBS+  (b). The average lengths and standard deviations97 3  ±  51 nm for (a) and 958  ±  53 nm or  b) .

We found a greater prevalence of the anomalous DNA in longermolecules such  as  lambda DNA than  in  shorter linearizedplasmids and the least amount in the closed circular plasmid form.The lengths of straightened lambda fragments were not measuredsince many fragments are produced in a partial £coRI digestion

and there would be no way of knowing the relative lengths ofthe straightened and the relaxed fragments. Whole lambda DNAmolecules were also found to be straightened in sections ratherthan over their entirety. Because of the surface coverage andconfusion at crossover points it was not possible to measure andcompare partially straightened molecules to relaxed lambda DNA.

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 withSma\  (b) the elongated strand was 78% longer than the relaxed linear DNAmolecules. Note that relaxed unstretched molecules of DNA appear to cross overstretched strandi.

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Nucleic Acids Research, 1 994, Vol. 22, No. 20   4227

Repulsion between DNA strands

An interesting feature of our preparations with moderate surfacecoverage is that the relaxed strands very rarely cross each other.Figure 4a shows AFM images of lambda DNA fragmentsprepared by critical point drying where a few of the fragmentsappear to be stretched. The relaxed DNA strands appear to avoidcrossing each other but the anomalous strands intersect the relaxedmolecules in an unfettered fashion. Figure 4b shows a typicalAFM image of circular pBS+  where none of the DNA strandscross each other. Th e anomalous strands do not appear to followthis rule; they cross the relaxed strands freely.

a

DISCUSSION

Increases in DNA lengths of up to 80% may appear to beunrealistic and well above elastic limits. Recent elastic studiesof DNA by Smith  et al.  (19) report no significant increases in

length of DNA in solution even when individual molecules werepulled by forces up to 10 n N. DNA is tightly coiled due tocoulombic, van der Waals, and molecular orbital forces.However, the deoxyribose-phosphate backbones are stronglyjoined by covalent bonds. It is conceivable that with sufficientexternal forces DNA can be stretched or uncoiled withoutbreaking the backbones. If DNA is modeled as a 2 nm wideladder with a helical repeat of 3.4 nm, an increase in length ofover a factor of two is possible by uncoiling the helix withoutbreaking the phosphate bonds (see Figure 5).

It may also be possible to stretch the DNA molecule withoutuncoiling. In this case the diameter of the helix must decrease.This would amount to 12% for a 30% stretch and 43% for an80%  stretch. Large changes in base pair distances are clearly

unreasonable, so that some uncoiling must occur for the longerelongations. Linear molecules would have the freedom rotatingto allow uncoiling and were found to be m ore elongated. C ircularmolecules had straightened sections but were less elongated. W especulate that some stretching and uncoiling may occur indifferent proportions for various conditions.

Deformation may take place during rinsing in a jet of waterwhere hydrodynamic forces pull on molecules that are not wellanchored to the substrate at all points along the molecular length.Though Shaiu  et al.  observed the aligning of DNA moleculesduring rinsing, they do not report any increase in DNA length(20).  In our images of anomalous molecules, the straightenedstrands were aligned generally in the same direction.

Unfortunately, sample alignment was not preserved during therinsing steps, so orientation with rinse direction cannot beverified. If they were elongated, one would think that possiblythe stored mechanical energy could be released by cutting thestrand into two pieces (this was achieved by increasing the contactforce 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 thesurface or tacked down at the point of dissection. Continuousattachment may also be indicated by the fact that we haveobserved some lambda fragments in the shape of a parabola ora semicircle.

[— 2.0—j

2 . 0  JT

Figure 4 .  AFM image of (a) fragments of lambda DNA and (b)  pB S+showing

possible electrostatic repulsion between the strands. The relaxed DNA strands

do not interact and remain separate from one another. In the lambda DNApreparation (a) some of the DNA strands appear to be straightened. Note thatunstretched relaxed DNA fragments freely cross the anomalous   DNA.

Figure 5.  A schematic showing the relationship between the length of normalB-form DNA, 3.4 nm helical repeat (10 bp), and the possible length of the

completely uncoiled molecule without breaking the phosphate linkages. Assuminga helical diameter of 2.0 nm, the maximum geometric extension would be 7.1/3.4or 2.1 times the normal length.

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4228 Nucleic Acids Research, 1994, Vol. 22, No. 20

The widths of DNA observed by AFM may be modified byadsorbed salt. When imaging was done at higher humidity( > 50%), apparent widths of relaxed DN A w ere found to increaseas much as 100% possibly due to the hygroscopic nature of buffersalt around the DNA strands (16). However, the anomalous DNA

did not show any appreciable increase in width at higher relativehumidity. Also, if die stretched DNA has a reduced layer of salt,its linear charge density may be different. This charge shouldalso be affected by the lengthening process  itself Since AFMtip deflection is due to a number of forces acting on the tipincluding electrostatic interaction, changes in the dipoleinteraction could contribute to the observed heights, especiallyfor DNA because of its small diameter and high linear chargedensity.

The decrease in height of DNA was found to depend on theextent of lengthening, with molecules whose contour lengthsshowed only slight lengthening being less affected than moleculesthat were highly lengthened. For example, the molecules thatare 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 andrelaxed regions. A gradient in height can also be observed inthe areas of transition in the same molecules (Figure lb).

The fact that relaxed DNA strands very rarely cross indicatesthat some repulsive force between the strands is operating as theDN A is being deposited. Repulsive forces acting after the surfacehas been covered would leave some strands crossing and in anunstable 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), theinteraction 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 nosignificant interaction between them, at least when the DNA isbeing deposited. If both types were laid down simultaneously,this would indicate that the anomalous DNA is electrostaticallyneutral. We speculate that the two strands are electrostaticallydifferent, and that the anomalo us DN A has lost its net charg e.

REFERENCES

1.  Thundat, T., Allison, D.P., W armack, 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., D elaine, E., Bustamante,C , (1992)  Ultramicroscopy  4 2 - 4 4 1243-1249.

3.  Henderson, E., (1992)  Nucleic Acids Res.  21 4 4 5 - 4 4 7 .4.  Bustamante, C , V esenka, J., Tang, C.L ., Rees, W., Gu thol d,M , Keller,R., (1992)  Biochem.  3 1 , 2 2 - 2 6 .

5.  H ansma, 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, 23—30.

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 , 8 2 4 - 8 2 8 .

8. Hansma, H.G., Bezannilla, M., Zenhausem, F ., Adrian, M., and Sinsheimer,R.L., (1993)  Nucleic Acids Res.  21 5 0 5 - 5 1 2 .

9. Lyubchenko, Y.L , Jacobs, B.L., and Lindsay, S.M., (1992) N ucleic Acids

Res.  20 3983-3986 .

10.  Thundat, T., Allison, D.P., Warmack, R.J., and Ferrell, T.L., (1992)Ultramicroscopy  4 2 - 4 4 1 0  - 1 1 0 6 .

11.  Allen, M.J., H ud, N.V ., Balooch, M. , Tench, R.J., Siekhaus, W. J., and

Ballhorn, R., (1992)  Ultramicroscopy  4 2 - 4 4 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. 5 0 157-170 .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 6 3 0 - 6 3 5 .17.  Chen, G.Y ., Warmack, R.J., Thundat, T., A llison, D.P., H uang, 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)  cience 258 1122-1126.20 .  Shaiu, W .L ., Larson. D.D ., Vesenka, J., and Henderson, E., (1993) Nucleic

Acid Res.  21 9 9 - 1 0 3 .

CONCLUSION

Unusual strands frequently appear in AFM images of plasmidand lambda DNA that have been adsorbed onto mica, vigorouslyrinsed, and dried. These strands differ from the usual relaxedstrands in several ways. Their lengths are up to about 80% longer

and their heights are about 50% less than relaxed strands. Theseobservations are consistent w ith stretching and uncoiling of theDNA helix possibly induced by rinsing followed by pinning tothe mica surface. Whereas the relaxed DNA strands appear tohave a linear charge density that prevents them from crossingeach other, the anomalous strands cross freely.

ACKNOWLEDGEMENTS

W e would like to thank M.C.R orv ik, K.B.Jacobson, J. Vesenkaand B.Hingerty for many helpful comments, and D.Englehartfor measuring apparent DNA lengths. This research wassponsored by the Office of Health and Environmental Research,

US Department of Energy under contract DE-AC05-84OR21400with Martin Marietta Energy- Systems, Inc.

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