Mechanical transition in a highly stretched and torsionally constrained DNA

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Mechanical transition in a highly stretched and torsionally constrained DNA

Janusz Strzelecki, Lukasz Peplowski, Robert Lenartowski, Wieslaw Nowak, and Aleksander Balter

Phys. Rev. E 89, 020701(R) – Published 27 February 2014

Abstract

We show results of our high force (up to 1.8 nN) atomic force microscopy 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 1 nN. This transition 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 the backbones. These results broaden our understanding of the fundamental aspects of DNA nanomechanics.

Received 27 September 2012

DOI:https://doi.org/10.1103/PhysRevE.89.020701

Authors & Affiliations

Janusz Strzelecki^1,*, Lukasz Peplowski¹, Robert Lenartowski², Wieslaw Nowak¹, and Aleksander Balter¹

¹Institute of Physics, Faculty of Physics, Astronomy and Informatics, Nicolaus Copernicus University, Grudziądzka 5, 87-100 Toruń, Poland
²Faculty of Biology and Environment Protection, Laboratory of Isotope and Instrumental Analysis, Lwowska 1, 87-100 Toruń, Poland

^*Corresponding author: jast@fizyka.umk.pl

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Vol. 89, Iss. 2 — February 2014

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Images

Figure 1
Stretching nonspecifically attached dsDNA using AFM force spectroscopy. An untreated AFM tip was used to pick and stretch the 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.
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Figure 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).
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Figure 3
The third plateau is present in a repeatedly stretched DNA molecule. The plateau was observed in a series of multiple stretchings 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).
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Figure 4
Normalization of ten 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 the overstretching and melting transition areas (a). A dependence of the plateau length on the molecule contour length [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 a 0.015 nm increase in length per base pair due to the 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.
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Figure 5
Lack of the third plateau in torsionally relaxed DNA molecules. The figure shows a set of six curves resulting from the stretching of unconstrained molecules, with the extension normalized for a force of 1100 pN. The plateau which was present in the constrained DNA is not observed here.
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Figure 6
SMD simulations of highly stretched DNA show a possible mechanism of the transition. The figure shows curves acquired from two 10 ns (gray) and one 100 ns (pink) SMD simulations of poly(dA-dT) poly(dA-dT) and two 10 ns (green) and 100 ns (blue) poly(dG-dC) poly(dG-dC). The rotation of the molecule during stretching was prevented. An experimental force curve (black) is included as a reference. The extension is calculated with 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 the backbones. The transition can thus be attributed to the transition into this form.
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