3rd Plateau DNA Final Draft

7/25/2019 3rd Plateau DNA Final Draft

1/17

1

Mechanical transition in a highly stretched and torsionally constrained DNA.

Janusz Strzelecki1, ukasz Pepowski1, Robert Lenartowski2, WiesawNowak1,

Aleksander Balter1

1 Institute of Physics, Nicolaus Copernicus University, Grudzidzka 5, 87-100 Toru,

Poland

2 Faculty of Biology and Environment Protection, Laboratory of Isotope and

Instrumental Analysis, Lwowska 1, 87-100 Toru, Poland

(Received

ABSTRACT

We show results of our high force (up to 1.8 nN) AFM force

spectroscopy measurements of a double stranded DNA. We have found that

the force spectra of torsionally constrained molecules display a small

plateau occurring at a force of approximately 1nN. This transition, not

reported before, is absent in molecules with rotational freedom. Based on

all-atom molecular dynamics simulations, we suggest that this plateau is a

result of reducing the diameter of a double helix through extreme

stretching. The simulation suggests that the molecule is forced into a form

resembling an underwound P-DNA, with bases protruding outside of

backbones. These results broaden our understanding of fundamental aspects

of DNA nanomechanics.

PACS numbers 87.15.La 87.14.gk 87.64.Dz


2/17

2

DNA is a key molecule of life and provides great hopes as a bionanotechnology

material [1]. Thus, since the very beginning of a single molecule force spectroscopy [2] this

all important biopolymer remains within focus of extensive nanomechanical research. DNA

was stretched and twisted in experiments involving optical/magnetic tweezers and atomic

force microscopy (AFM) [3-6]. Those experiments revealed that the DNA double helix can

be overstretched up to 70% of contour length when pulled. This transition manifests as a

force-specific plateau on a force - distance curve [7-8]. In majority of experiments the ends of

double helix may rotate along a long axis of DNA and then the plateau is observed at 65 pN

[9-10]. In torsionally constrained setting, when such rotation is not possible, the force is 110

pN [7-8, 11-12]. A second plateau, marking the full strands melting, was encountered at

approximately 300 pN [13-15]. Despite significant research effort, very few experiments

explored the DNA mechanics beyond 500pN limit. Such extreme deformation of double

stranded DNA (dsDNA) should be feasible, as single stranded DNA (ssDNA) was stretched

with forces as large as 2nN [13, 16]. To date, only stretching torsionally unconstrained DNA

with forces up to 1 nN was reported [13]. A more detailed study of DNA mechanics in this

high force regime, including stretching of torsionally constrained molecules is still missing .

This regime is biologically important. A recent research [17-18] suggests that some damage

acquired by genetic material during abnormal mitosis can result from extreme stretching and

rupturing of DNA when the chromosomes are pulled apart. Such damage can cause mutations

and trigger tumor initiation and progression. Additionally, extracellular DNA that strengthens

bacterial biofilm [19] is also exposed to significant deforming forces by the environment.

Knowing the behavior of double helix at its mechanical limits is also important in case of its

potential applications as a bionanotechnology building block [1].


3/17

3

In this paper, we report an experimental evaluation of dsDNA stretched up to nN

force range. We observed for the first time, to the best of our knowledge, a mechanical

transition exhibited as a third plateau in force spectra of torsionally constrained molecules. A

mechanism of the plateau origin supported by Steered Molecular Dynamics (SMD)

simulations is proposed.

A simple, and yet a very effective method of nonspecific molecule picking with an

untreated AFM tip allowed us to achieve a stretching force close to 2nN. We used the pUC18

plasmid (Fermentas, SD0051), linearized with the EcoRI enzyme (Fermentas, ER0271). To

check a possible influence of the DNA base pair sequence and a molecule length on the

observed effect, a lambda phage (Fermentas, SD0011) was studied as well using the same

protocol. SMD simulations have been performed using NAMD 2.8 code [20] and all-atom

CHARMM27 force field [21]. Simulations mimicked topologies of our AFM force

spectroscopy experiment - dragged atoms had constraints in all directions except the helical

axis, thus preventing rotation during stretching. A control set of simulations was also made,

with the pulling carbon atoms unconstrained and a free rotation around the helical axis [22].

In the majority of cases molecules picked with an AFM tip detached and were lost at

stretching forces below 0.5 nN. Nevertheless, patient molecule fishing allowed us to record

48 force curves (each resulting from different molecule) above this limit, with detachment

force beyond 1 nN (a maximum observed stretching force was 1.8 nN). Force curves obtained

showed characteristics either of torsionally constrained (rounded onset of overstretching

transition at 110pN, no hysteresis upon relaxation) or unconstrained DNA stretching (sharp

onset of overstretching at 65pN, significant hysteresis upon relaxation). Apart from the well

known overstretching and melting transitions also a third, never previously reported plateau


4/17

4

was present, but only if the stretched molecule was torsionally constrained (Fig. 1). This

feature appeared at very high extensions, when the molecule was stretched approximately 2.3

times its contour length and for forces approaching 1 nN. As can be seen in Fig. 1, this

plateau is very small when compared to the overstretching and melting transitions.

Force spectrograms of molecules that did not detach during stretching show that the

transition is reversible (Fig. 2(a)) and is present both in the stretching and relaxing curves

similarly to the overstretching and melting. This plateau was observed in each constrained

DNA force curve, obtained in independent experiments on different samples and in the

molecules of different length (Fig. 2(b)), provided the stretching force was large enough.

Repetitive stretching of the same molecule also marked its presence in each force

spectrogram (Fig. 3(a)). Additionally, when a portion of a stretched molecule detached and

was stretched again the transition appeared each time (Fig. 3(b)). This third plateau cannot

thus be considered just a minor detachment event or an instrumental artifact. In our opinion it

is an integral feature of DNA mechanics and reflects a forced conformational change within

the highly stretched double helix itself.

To directly compare data from different molecules we employed extension normalization

procedures [23] for 10 curves with molecular extension at 1100 pN (Fig. 4(a)). In order to

account for the influence of base pair sequence and a significant variation in the contour

length, additional measurements with the lambda phage DNA were made and force curves

were analyzed along with the curves obtained or pUC18 plasmid DNA. While analyzed

curves overlap perfectly within the overstretching and melting plateaus region, a significant

variation can be seen in the third plateau. It was observed for a certain range of forces (820-

1020 pN) rather than a specific value, as in the case of overstretching transition (Fig. 4(b)).


5/17

5

This force value does not seem to depend on the length of the stretched molecule. The

extension resulting from this transition is, on the other hand, clearly proportional to the

contour length (Fig. 4(a) inset). A rough estimate with a linear fit yields a 0.015 nm extension

per single base pair resulting from this plateau.

The third plateau never appeared when the stretched molecule was unconstrained.

This can be clearly seen on Fig 5, where normalization was made for force curves showing

characteristics of resulting from molecules with a free rotation. This implies that the origin of

this transition is inherently connected with the DNA torsional constrain.

In order to explain the mechanism behind this plateau we performed SMD simulations

for both constrained and unconstrained dsDNA.Those simulations show that when the DNA

cannot unwind, the stretched double helix reduces its diameter, forcing both strands into

contact. As a result, stacking is disrupted and bases start protruding outwards (Fig. 6). The

molecule adopts a shape similar to the previously reported P-DNA [24]. However, the

structure obtained through extreme stretching is underwound in comparison to the P-DNA

created by the extreme overtwisting with magnetic tweezers. Conversion of SMD force vs

extension curves relative to the contour length allowed for comparison with AFM force

spectroscopy data. We can see that the plateau coincides well with the transition to the

underwound P-DNA. This leads to conclusion that this origin of the third plateau is a sound

hypothesis.

SMD with a free rotation of stretched molecule leads to formation of the Zip-DNA

form, with an interchangeable base stacking, reported previously [25]. This explains the lack

of the third plateau in unconstrained molecules, as stretching induced helix diameter


6/17

6

reduction cannot take place in that case. Additionally, we can expect the force necessary to

cause the diameter reduction would depend on the arrangement of the DNA helix. On the

other hand, in AFM experiment with nonspecific molecule attachment a variation in pulling

angle or in separation of DNA strands is very likely. Those conditions can explain the fact

that the plateau did appear within a certain range of forces rather than for a specific one.

We did a rough estimate to test this model further. A height of a single helical pitch is

equal to , with lbeing the helix length and r the helix radius. With 38 bp S-

DNA present after overstretching a helix length would be 26.6 nm. Based on AFM imaging

of stretched DNA [26] we can assume that before the transition the helix radius is

approximately 0.6 nm. Reducing the helix radius by 0.25 nm, which would correspond to the

average size (0.5 nm) of a single pyrimidine and purine base would result in elongation of

helical pitch by 0.18 nm and 0.0047 nm per single base pair. The extension of 0.015 nm per

single base pair obtained from the linear fit from the inset on Fig 4 is approximately three

times smaller. However, experimental results indicate that 1/5 of overstretched torsionally

constrained DNA has P-DNA form [12], thus 1/5 of base pairs could not contribute to the

extension through the diameter reduction. Additionally, further bases protruding outside

backbones appear beyond overstretching transition [27] due to melting. Thus, when

comparing the extension per single base pair obtained from our experiment with a proposed

model, the actual value should not be calculated over all base pairs, but adjusted accordingly

at the very least by a factor of 0.8. Considering this, we can find a good agreement between

our results and this simple geometrical estimate.

In summary, we report for the first time the presence of a mechanical transition in a

highly stretched DNA with a torsional constraint. Based on SMD simulations we postulate


7/17

7

that the plateau observed in force spectra is caused by the double helix diameter reduction of

the strained molecule, leading to formation of structure similar to the underwound P-DNA.

Our results give a new insight into the understanding of DNA mechanics. The DNA proves to

be a remarkably resilient molecule. As long as the double helix does not unwind, it is not

completely denatured until a force approaching 1 nN is reached. Even after such extreme

stretching, this molecule may easily return to its relaxed conformation. Additionally, if the

hypothesis of the forced transition to an underwound P-DNA is correct, then a new

mechanism, besidest the full separation of strands or overtwisting, to expose nucleobases

seems to be possible. Thus, strong mechanical deformation could serve as a means to provide

an access to the genetic sequence. A similar mechanism of opening binding sites in globular

proteins through the mechanical unfolding has been postulated [28].

ACKNOWLEDGEMENTS

We thank Prof. Piotr E. Marszalek for stimulating our work in single molecule force

spectroscopy and Prof. Richard Lavery for DNA pdb files.

Janusz Strzelecki acknowledges grant Krok w przyszo(I and III edition) from Marszaek

of Kujawsko-Pomorskie Voivodeship and EU.

[1] A. V. Pinheiro, D. Han, W. M. Shih, and H. Yan, Nature nanotechnology (2011).

[2] S. B. Smith, L. Finzi, and C. Bustamante, Science 258, 1122 (1992).

[3] C. Bustamante, S. B. Smith, J. Liphardt, and D. Smith, Current Opinion in Structural

Biology 10, 279 (2000).

[4] C. Bustamante, Z. Bryant, and S. B. Smith, Nature (London), 423 (2003).

[5] J. F. Marko and S. Cocco, Phys. World 16, 37 (2003).

[6] C. Prvost, M. Takahashi, and R. Lavery, ChemPhysChem 10, 1399 (2009).


8/17

8

[7] S. B. Smith, Y. Cui, and C. Bustamante, Science 271, 795 (1996).

[8] P. Cluzel, A. Lebrun, C. Heller, R. Lavery, J. L. Viovy, D. Chatenay, and F. Caron,

Science 271, 792 (1996).

[9] N. Bosaeus, A. H. El-Sagheer, T. Brown, S. B. Smith, B. kerman, C. Bustamante,

and B. Nordn, Proceedings of the National Academy of Sciences 109, 15179 (2012).

[10] X. Zhang, H. Chen, S. Le, I. Rouzina, P. S. Doyle, and J. Yan, Proceedings of the

National Academy of Sciences 110, 3865 (2013).

[11] D. H. Paik and T. T. Perkins, Journal of the American Chemical Society (2011).

[12] J. Leger, G. Romano, A. Sarkar, J. Robert, L. Bourdieu, D. Chatenay, and J. Marko,

Physical review letters 83, 1066 (1999).

[13] M. Rief, H. Clausen-Schaumann, and H. E. Gaub, Nature Structural Biology 6, 346

(1999).

[14] H. Clausen-Schaumann, M. Rief, C. Tolksdorf, and H. E. Gaub, Biophysical Journal

78, 1997 (2000).

[15] C. P. Calderon, W. H. Chen, K. J. Lin, N. C. Harris, and C. H. Kiang, Journal of

Physics: Condensed Matter 21, 034114 (2009).

[16] T. Hugel, M. Rief, M. Seitz, H. E. Gaub, and R. R. Netz, Physical Review Letters 94,

48301 (2005).

[17] A. A. Guerrero, M. C. Gamero, V. Trachana, A. Ftterer, C. Pacios-Bras, N. P. Daz-

Concha, J. C. Cigudosa, C. Martnez-A, and K. H. M. Van Wely, Proceedings of the

National Academy of Sciences 107, 4159 (2010).

[18] N. J. Ganem and D. Pellman, The Journal of Cell Biology 199, 871 (2012).

[19] W. Huet al., PloS one 7, e51905 (2012).

[20] J. C. Phillipset al., Journal of Computational Chemistry 26, 1781 (2005).

[21] A. D. MacKerell Jret al., The Journal of Physical Chemistry B 102, 3586 (1998).

[22] See Supplemental Material for the AFM force spectroscopy experiment procedure and

SMD simulations details.

[23] M. Rabbi and P. E. Marszalek, Cold Spring Harbor Protocols 2007, pdb. prot4900

(2007).

[24] J. Allemand, D. Bensimon, R. Lavery, and V. Croquette, Proceedings of the National

Academy of Sciences 95, 14152 (1998).

[25] A. Balaeff, S. L. Craig, and D. N. Beratan, The Journal of Physical Chemistry A

(2011).

[26] M. Maaloum, A. Beker, and P. Muller, Physical Review E 83, 031903 (2011).


9/17

9

[27] J. Van Mameren, P. Gross, G. Farge, P. Hooijman, M. Modesti, M. Falkenberg, G. J.

L. Wuite, and E. J. G. Peterman, Proceedings of the National Academy of Sciences

106, 18231 (2009).

[28] R. Perez-Jimenez, A. P. Wiita, D. Rodriguez-Larrea, P. Kosuri, J. A. Gavira, J. M.

Sanchez-Ruiz, and J. M. Fernandez, Journal of Biological Chemistry 283, 27121

(2008).


10/17

10

FIGURE CAPTIONS

FIG. 1. Stretching non-specifically attached dsDNA using AFM force spectroscopy.

Untreated AFM tip was used to pick and stretch DNA molecules with a force reaching almost

2 nN. Representative force curves obtained for molecules of similar length but different in

torsional arrangement are shown. A small third plateau, absent in a torsionally relaxed DNA

is visible in a constrained molecule.

FIG. 2. A third plateau observed in a highly stretched constrained DNA.The transition

underlying this event is reversible, as it appears both during the stretching (upper black) and

the relaxation (lower red) of a single DNA molecule (a). A magnified fragment of a force

curve, showing the third plateau in detail is displayed in the inset. This plateau was observed

each time a force exceeding 800 pN was achieved, as can be seen in three representative

curves obtained for different molecules on different samples, with significant variation in

contour length (b).

FIG. 3 The third plateau is present in repeatedly stretched DNA molecule.The plateau

was observed in a series of multiple stretching of the same DNA molecule (a). Two curves

(upper black stretching, lower red relaxation) that show this transition both before and after a

minor detachment of a stretched molecule are also shown (b).

FIG. 4 Normalization of 10 force curves showing the third plateau and a histogram of

transition forces.Curves were normalized for the force of 1100 pN [9 pUC18 (black) and 1

lambda phage (bright green)]. A full overlap can be seen in overstretching and melting

transition areas (a). A dependence of plateau length on the molecule contour length


11/17

11

[expressed in number of base pairs (bp)] is shown in the inset [pUC18 (black square) and 1

lambda phage (bright green diamonds)]. A linear fit to this data (red solid line) gives an

estimate of 0.015 nm increase in length per base pair due to transition. This plateau appears

for a certain range of forces (820 -1020 pN) (b) instead of a specific one. A molecule length

does not seem to influence the value of those forces.

FIG. 5. Lack ofthe third plateau in torsionally relaxed DNA molecules.The figure shows

a set of 6 curves resulting from the stretching of unconstrained molecules, with the extension

normalized for force of 1100 pN. The plateau which was present in the constrained DNA is

not observed here.

FIG. 6. SMD simulations of highly stretched DNA show a possible mechanism of the

transition.The figure shows curves acquired from two 10 ns (grey) and one 100 ns (pink)

SMD simulations of poly(dA-dT) poly(dA-dT) and two 10 ns (green) and 100ns (blue)

poly(dG-dC) poly(dG-dC). The rotation of molecule during stretching was prevented. An

experimental force curve (black) is included as a reference. The extension is calculated in

reference to the initial B-DNA form length. Snapshots from different phases of the

simulation, marked by orange dots are also shown. A constrained stretched molecule is

forced to reduce its diameter as the extension increases and the base pairing and stacking is

disrupted. At the extension coincident with the plateau a structure resembling an underwound

P-DNA is formed, with base pairs protruding outside of backbones. The transition can thus be

attributed to the transition into this new form.


12/17

12

FIG. 1

Strzelecki


13/17

13

FIG. 2

Strzelecki


14/17

14

FIG. 3

Strzelecki


15/17

15

FIG. 4

Strzelecki


16/17

16

FIG. 5

Strzelecki


17/17

17

FIG. 6

Strzelecki

3rd Plateau DNA Final Draft

Documents

Transcript of 3rd Plateau DNA Final Draft