Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers

Kriegel, Franziska; Ermann, Niklas; Lipfert, Jan

doi:10.1016/j.jsb.2016.06.022

Cited by 87 publications

(100 citation statements)

References 145 publications

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“…Positive supercoiling occurs up to forces of ∼6 pN. For the highest measured force, 6.5 pN, no buckling occurs at all, instead the DNA molecules undergo a transition from B- to P-DNA at ∼35 pN·nm (10,67,74) (Figure 2E). …”

Section: Resultsmentioning

confidence: 97%

“…At length scales significantly longer than 1 bp, DNA is commonly modeled as an isotropic elastic rod, with a bending persistence length A , stretch or Young's modulus B , torsional stiffness C and twist-stretch coupling D (9,10). All four elastic constants have been determined for DNA in vitro in a series of ingenious single-molecule manipulation measurements (11–20).…”

Section: Introductionmentioning

confidence: 99%

“…Using this experimental approach, we determine the torsional stiffness of DNA as a function of applied stretching force and salt concentration. Our mMTT instrument is an extension of conventional MT that have proven to be a powerful tool to investigate the torsional properties of nucleic acids (10,52,53). In MT and mMTT, molecules of interest are tethered between a flow cell surface and magnetic particles (Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers

Kriegel

Ermann

Forbes

et al. 2017

Nucleic Acids Research

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The mechanical properties of DNA fundamentally constrain and enable the storage and transmission of genetic information and its use in DNA nanotechnology. Many properties of DNA depend on the ionic environment due to its highly charged backbone. In particular, both theoretical analyses and direct single-molecule experiments have shown its bending stiffness to depend on salt concentration. In contrast, the salt-dependence of the twist stiffness of DNA is much less explored. Here, we employ optimized multiplexed magnetic torque tweezers to study the torsional stiffness of DNA under varying salt conditions as a function of stretching force. At low forces (<3 pN), the effective torsional stiffness is ∼10% smaller for high salt conditions (500 mM NaCl or 10 mM MgCl2) compared to lower salt concentrations (20 mM NaCl and 100 mM NaCl). These differences, however, can be accounted for by taking into account the known salt dependence of the bending stiffness. In addition, the measured high-force (6.5 pN) torsional stiffness values of C = 103 ± 4 nm are identical, within experimental errors, for all tested salt concentration, suggesting that the intrinsic torsional stiffness of DNA does not depend on salt.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers

Kriegel

Ermann

Forbes

et al. 2017

Nucleic Acids Research

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“…A satisfactory explanation may involve differences in the atomic-scale structure that are beyond a continuum mechanics analysis (19,21).…”

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confidence: 99%

Understanding the mechanical response of double-stranded DNA and RNA under constant stretching forces using all-atom molecular dynamics

Marín-González

Vilhena

Pérez

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

152

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Multiple biological processes involve the stretching of nucleic acids (NAs). Stretching forces induce local changes in the molecule structure, inhibiting or promoting the binding of proteins, which ultimately affects their functionality. Understanding how a force induces changes in the structure of NAs at the atomic level is a challenge. Here, we use all-atom, microsecond-long molecular dynamics to simulate the structure of dsDNA and dsRNA subjected to stretching forces up to 20 pN. We determine all of the elastic constants of dsDNA and dsRNA and provide an explanation for three striking differences in the mechanical response of these two molecules: the threefold softer stretching constant obtained for dsRNA, the opposite twist-stretch coupling, and its nontrivial force dependence. The lower dsRNA stretching resistance is linked to its more open structure, whereas the opposite twist-stretch coupling of both molecules is due to the very different evolution of molecules' interstrand distance with the stretching force. A reduction of this distance leads to overwinding in dsDNA. In contrast, dsRNA is not able to reduce its interstrand distance and can only elongate by unwinding. Interstrand distance is directly correlated with the slide base-pair parameter and its different behavior in dsDNA and dsRNA traced down to changes in the sugar pucker angle of these NAs.

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“…By manipulation of microbeads, single-molecule force spectroscopy techniques revealed the mechanical properties of biomolecules such as DNA or RNA with unprecedented detail [2][3][4][5]. In addition, the interactions with proteins, like the DNA compaction by histones in eukaryotic chromatin [6][7][8][9][10][11] and prokaryotic architectural proteins [12][13][14][15][16], supercoiling [17][18][19][20], and repair processes [21][22][23] were extensively studied with magnetic tweezers (MT) or optical tweezers (OT), Acoustic Force Spectroscopy (AFS) [24][25][26] or tethered particle motion (TPM) [13,27,28]. These bead manipulation techniques have also been used to quantify the mechanical properties of other biological structures, such as extracellular protein collagen [29][30][31], or even entire cells [32].…”

Section: Introductionmentioning

confidence: 99%

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

Brouwer

Hermans

Noort

2019

Preprint

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Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method. Here, we introduce 3D phasor tracking, a fast and robust bead tracking algorithm with nanometric localization accuracy in arange of over 10 µm. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10.000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required. SignificanceMicrobeads are often used in biophysical single-molecule manipulation experiments and accurately tracking their position in 3 dimensions is key for quantitative analysis. Holographic imaging of these beads allows for multiplexing bead tracking but image analysis can be a limiting factor. Here we present a 3D tracking algorithm based on Fast Fourier Transforms that is fast, has nanometric precision, is robust against common artifacts and is accurate over 10's of micrometers. We show its real-time application for magnetic tweezers based force spectroscopy on more than 100 chromatin fibers in parallel, but we anticipate that many other bead-based biophysical essays can benefit from this simple and robust 3 phasor algorithm.

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Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers

Cited by 87 publications

References 145 publications

Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers

Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers

Understanding the mechanical response of double-stranded DNA and RNA under constant stretching forces using all-atom molecular dynamics

Multiplexed Nanometric 3D Tracking of Microbeads using a FFT-Phasor Algorithm

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