Quantitative Guidelines for Force Calibration through Spectral Analysis of Magnetic Tweezers Data

Velthuis, Aartjan J.W. te; Kerssemakers, Jacob; Lipfert, Jan; Dekker, Nynke H.

doi:10.1016/j.bpj.2010.06.008

Cited by 102 publications

(102 citation statements)

References 32 publications

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“…Using the ability of MTs to exert precisely calibrated stretching forces (18,19) (Materials and Methods and SI Appendix, Fig. S1), we first probed the forceextension response of dsRNA.…”

Section: Resultsmentioning

confidence: 99%

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Lipfert

Skinner

Keegstra

et al. 2014

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

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175

258

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RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a forcetorque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level.RNA | nucleic acids | magnetic tweezers | force | torque

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“…Using the ability of MTs to exert precisely calibrated stretching forces (18,19) (Materials and Methods and SI Appendix, Fig. S1), we first probed the forceextension response of dsRNA.…”

Section: Resultsmentioning

confidence: 99%

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Lipfert

Skinner

Keegstra

et al. 2014

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

Self Cite

175

258

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“…We developed a magnetic tweezer assay (17)(18)(19) that allowed us to modulate the force applied to individual DNA molecules in the presence of Dps (Fig. S1C).…”

Section: Significancementioning

confidence: 99%

Hysteresis in DNA compaction by Dps is described by an Ising model

Vtyurina

Dulin

Docter

et al. 2016

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

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In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNAbinding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.DNA condensation | Dps | cooperativity | hysteresis | Ising model P urified DNA behaves as an entropic spring with a radius of gyration that scales as a function of the contour length (1). In contrast, DNA in vivo is highly organized and condensed. In bacteria, this condensation is caused by nucleoid-associated proteins (NAPs) that collectively shape the chromosome (2, 3). NAPs are capable of binding genomic DNA and in doing so alter its shape, control the transcriptional expression of genes, and remodel the structure of the nucleoid in response to external stimuli (2, 3).DNA-binding protein from starved cells (Dps) is an NAP structurally related to ferritins and associated with the response to stress. Dps is highly expressed in stationary phase (4-7) and is also involved in the cellular response to oxidative (4, 8-10), UV (8, 11), thermal (8), and pH shocks (8). In addition, Dps has been implicated in biofilm formation and tolerance to bacteriophage attacks (12). Dps monomers have a molecular mass of 19 kDa and assemble into a dodecameric shell (Fig. S1A) (13). The resulting complex binds to both supercoiled and linear DNA to form a dense biocrystal structure (4,7,9,14).Although the crystal structure of the Dps dodecamer has been solved (13), no atomic-scale structure of Dps-DNA assemblies currently exists and little is known about complex formation. The affinity of Dps for DNA is very sensitive to buffer conditions. Like many DNA-binding proteins, Dps binds DNA more weakly in the presence of higher salt concentrations. Less typically, divalent cations such as Mg 2+ can substantially weaken the affinity of Dps for DNA (9, 15). It has been proposed that fluctuations in divalent cation concentrations act as a trigger for biocrystal as...

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“…Using a conventional MT (see Discussion) with appropriate field configuration ( Figure 1a) and both translational and rotational control of the magnet position, search for rotationally constrained DNA molecules in the flow cell. At pulling forces of ≥ 1 pN (consult references 4,19,20,28,29 regarding force calibration in magnetic tweezers), tethered beads can easily be distinguished from beads stuck to the surface of the bottom slide by their different heights in the focus. Whether a DNA molecule is rotationally constrained can be assessed by introducing 20-30 turns of the magnets at a force of ≈ 0.25 pN: here, the tether length should decrease by 0.4-0.5 μm.…”

Section: Measurements On a Single Dna Molecule In The Conventional Mamentioning

confidence: 99%

Magnetic Tweezers for the Measurement of Twist and Torque

Lipfert

Lee

Ordu

et al. 2014

JoVE

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Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a "conventional" magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the "conventional" magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

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Quantitative Guidelines for Force Calibration through Spectral Analysis of Magnetic Tweezers Data

Cited by 102 publications

References 32 publications

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

Hysteresis in DNA compaction by Dps is described by an Ising model

Magnetic Tweezers for the Measurement of Twist and Torque

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