Multiplexed single-molecule measurements with magnetic tweezers

Ribeck, Noah; Saleh, Omar A.

doi:10.1063/1.2981687

Cited by 151 publications

(165 citation statements)

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“…Next, we demonstrate that although our force control is at the singlemolecule level, our folding scheme can fold multiple DNA nanostructures in a parallel manner. Among the tools for singlemolecule force spectroscopy, magnetic tweezers uniquely permits parallel manipulation of many single molecules 31,35 , because the gradient of magnetic field is largely constant over a long length scale, for example, a millimetre scale for our magnet configuration 33 (Supplementary Fig. 10).…”

Section: Resultsmentioning

confidence: 99%

“…1c). Magnetic tweezers can manipulate single DNA molecules with pico-newton force while probing their end-to-end distances with a nanometre resolution [17][18][19][20]26,[31][32][33][34][35][36] . In addition, different from other methods for single-molecule force spectroscopy, magnetic tweezers inherently permits multiplexed manipulation, which allows us to fold DNA nanostructures in a highly parallel manner in one experiment (see below).…”

Section: Resultsmentioning

confidence: 99%

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Programmed folding of DNA origami structures through single-molecule force control

et al. 2014

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Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is well defined and free from kinetic traps, enabling us to complete folding within 10 min. We also demonstrate parallel folding of DNA nanostructures through multiplexed manipulation of the scaffold DNAs. Our results suggest a path towards programmability of the folding pathway of DNA nanostructures.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Programmed folding of DNA origami structures through single-molecule force control

et al. 2014

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“…The elasticity of 1-6 µm intrinsically single-stranded NAs was measured using magnetic tweezers (23,24), in which the molecule is attached, at one end, to the substrate and, at the other end, to a paramagnetic bead. A magneticfield gradient exerts a force on the bead, and the resulting molecular extension is measured.…”

Section: Resultsmentioning

confidence: 99%

“…The molecular elasticity of the ssNA samples was measured using single-molecule magnetic tweezers force spectroscopy (23,24). The molecule under study was attached, at one end, to the surface of a glass coverslip and, at the other end, to a micrometer-scale paramagnetic bead (2.8 µm Dynabeads M-280).…”

Section: Methodsmentioning

confidence: 99%

Single-stranded nucleic acid elasticity arises from internal electrostatic tension

Jacobson

McIntosh

Stevens

et al. 2017

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

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Understanding of the conformational ensemble of flexible polyelectrolytes, such as single-stranded nucleic acids (ssNAs), is complicated by the interplay of chain backbone entropy and saltdependent electrostatic repulsions. Molecular elasticity measurements are sensitive probes of the statistical conformation of polymers and have elucidated ssNA conformation at low force, where electrostatic repulsion leads to a strong excluded volume effect, and at high force, where details of the backbone structure become important. Here, we report measurements of ssDNA and ssRNA elasticity in the intermediate-force regime, corresponding to 5-to 100-pN forces and 50-85% extension. These data are explained by a modified wormlike chain model incorporating an internal electrostatic tension. Fits to the elastic data show that the internal tension decreases with salt, from >5 pN under 5 mM ionic strength to near zero at 1 M. This decrease is quantitatively described by an analytical model of electrostatic screening that ascribes to the polymer an effective charge density that is independent of force and salt. Our results thus connect microscopic chain physics to elasticity and structure at intermediate scales and provide a framework for understanding flexible polyelectrolyte elasticity across a broad range of relative extensions.single-stranded nucleic acids | flexible polyelectrolytes | force spectroscopy | electrostatics S ingle-stranded nucleic acids (ssNAs) occur in many biological processes, such as RNA folding (1, 2) and DNA replication (3-5), generally in disordered conformations that fluctuate between various structures. These fluctuations create a significant entropic elasticity; thus, direct measurements of molecular elasticity (extension, X , as a function of applied force, fapp) constitute a powerful tool for studying the ssNA structural ensemble (6). In particular, measurements at a particular fapp are sensitive to the conformation within the corresponding tensile length, kB T /fapp (to within a scaling factor), where kB T is the thermal energy (7).ssNA structure-and that of flexible polyelectrolytes more generally-is complicated by the strong negative charge of the molecule. Multiple electrostatic and structural length scales share a similar magnitude (roughly 1 nm), disallowing models that consider only electrostatics or only backbone structure. In an uncharged flexible chain, the key structural scale is the persistence length, lp, defining the distance beyond which thermal fluctuations strongly bend the polymer. Electrostatic interactions introduce several competing length scales: the distance, b, between charged phosphates along the ssNA backbone; the distance over which electrostatic fields are screened in salty solution (the Debye length, κ −1 ); and the distance at which interactions between elementary charges in water have energy kB T (the Bjerrum length, lB ).Prior studies have elucidated ssNA elastic behavior in the lowand high-force limits. At low forces, corresponding to tensile lengths larger than κ −1 (i....

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“…Bead positions were tracked by placing the flow cell in an inverted light microscope, imaging the beads onto a 60 Hz CCD camera, and calculating the bead position with nanometer accuracy using existing imageanalysis routines (31,32). To remove common-mode drift, both experimental (gel-attached) beads, and reference beads (attached to the glass surface) were tracked; the reported trajectories correspond to the difference between the experimental and reference traces.…”

Section: Methodsmentioning

confidence: 99%

Active, motor-driven mechanics in a DNA gel

Bertrand

Fygenson

Saleh

2012

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

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Cells are capable of a variety of dramatic stimuli-responsive mechanical behaviors. These capabilities are enabled by the pervading cytoskeletal network, an active gel composed of structural filaments (e.g., actin) that are acted upon by motor proteins (e.g., myosin). Here, we describe the synthesis and characterization of an active gel using noncytoskeletal components. We use methods of base-pair-templated DNA self assembly to create a hybrid DNA gel containing stiff tubes and flexible linkers. We then activate the gel by adding the motor FtsK50C, a construct derived from the bacterial protein FtsK that, in vitro, has a strong and processive DNA contraction activity. The motors stiffen the gel and create stochastic contractile events that affect the positions of attached beads. We quantify the fluctuations of the beads and show that they are comparable both to measurements of cytoskeletal systems and to theoretical predictions for active gels. Thus, we present a DNA-based active gel whose behavior highlights the universal aspects of nonequilibrium, motor-driven networks.L iving cells display a range of interesting mechanical activities, including the ability to change shape, to translocate, and to apply stress to their surroundings. All of these activities can be stimulated by external mechanical or chemical signals. Some do not rely on a genetic response, but rather a response at the level of the cytoskeleton, the primary mechanical system of the cell (1, 2). This suggests that some of the cell's mechanical activities can be reproduced by a relatively simple artificial soft-matter system that lacks genetic control. Such a system would be useful both as a model for testing the physical principles underlying cytoskeletal behavior and as a material with potential technological applications.To design such a system, we must consider the key components of the cytoskeleton itself: Many cellular mechanical activities are due to a network of actin filaments that gains an active, nonequilibrium character through internal stresses generated by myosin motor proteins. These motors transduce chemical energy (through ATP hydrolysis) into mechanical work. In both muscle and nonmuscle cells, the main mechanical role of myosin is to contract the filaments, creating stress that propagates through the actin gel (3). This leads to a stiffening of the cell (4) along with enhanced mechanical fluctuations (5). These two behaviors have also been seen in reconstituted networks that only contain myosin and cross-linked actin (6-9), supporting the notion that these two components are the minimum needed to recapitulate active cell mechanics.What other motors and filaments can be used to form a gel with active mechanics? There are many biomolecular and synthetic fibers that can match the structural stiffness and cross-linking capabilities of actin (if not its active polymerization traits). The motor proteins are not so easily replaced, as can be judged by the limited capabilities of synthetic molecular motors in comparison to their biologi...

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Multiplexed single-molecule measurements with magnetic tweezers

Cited by 151 publications

References 14 publications

Programmed folding of DNA origami structures through single-molecule force control

Programmed folding of DNA origami structures through single-molecule force control

Single-stranded nucleic acid elasticity arises from internal electrostatic tension

Active, motor-driven mechanics in a DNA gel

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