Dataset Information

Correction-free force calibration for magnetic tweezers experiments.

ABSTRACT: Magnetic tweezers are a powerful technique to perform high-throughput and high-resolution force spectroscopy experiments at the single-molecule level. The camera-based detection of magnetic tweezers enables the observation of hundreds of magnetic beads in parallel, and therefore the characterization of the mechanochemical behavior of hundreds of nucleic acids and enzymes. However, magnetic tweezers experiments require an accurate force calibration to extract quantitative data, which is limited to low forces if the deleterious effect of the finite camera open shutter time (?_sh) is not corrected. Here, we provide a simple method to perform correction-free force calibration for high-throughput magnetic tweezers at low image acquisition frequency (f_ac). By significantly reducing ?_sh to at least 4-fold the characteristic times of the tethered magnetic bead, we accurately evaluated the variance of the magnetic bead position along the axis parallel to the magnetic field, estimating the force with a relative error of ~10% (standard deviation), being only limited by the bead-to-bead difference. We calibrated several magnets - magnetic beads configurations, covering a force range from ~50?fN to ~60?pN. In addition, for the presented configurations, we provide a table with the mathematical expressions that describe the force as a function of the magnets position.

SUBMITTER: Ostrofet E

PROVIDER: S-EPMC6206022 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Correction-free force calibration for magnetic tweezers experiments.

Ostrofet Eugen E Papini Flávia Stal FS Dulin David D

Scientific reports 20181029 1

Magnetic tweezers are a powerful technique to perform high-throughput and high-resolution force spectroscopy experiments at the single-molecule level. The camera-based detection of magnetic tweezers enables the observation of hundreds of magnetic beads in parallel, and therefore the characterization of the mechanochemical behavior of hundreds of nucleic acids and enzymes. However, magnetic tweezers experiments require an accurate force calibration to extract quantitative data, which is limited t ...[more]

PMID: 30374099

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Project description:Single-molecule tweezers measurements of double-stranded nucleic acids (dsDNA and dsRNA) provide unprecedented opportunities to dissect how these fundamental molecules respond to forces and torques analogous to those applied by topoisomerases, viral capsids, and other biological partners. However, tweezers data are still most commonly interpreted post facto in the framework of simple analytical models. Testing falsifiable predictions of state-of-the-art nucleic acid models would be more illuminating but has not been performed. Here we describe a blind challenge in which numerical predictions of nucleic acid mechanical properties were compared to experimental data obtained recently for dsRNA under applied force and torque. The predictions were enabled by the HelixMC package, first presented in this paper. HelixMC advances crystallography-derived base-pair level models (BPLMs) to simulate kilobase-length dsDNAs and dsRNAs under external forces and torques, including their global linking numbers. These calculations recovered the experimental bending persistence length of dsRNA within the error of the simulations and accurately predicted that dsRNA's "spring-like" conformation would give a two-fold decrease of stretch modulus relative to dsDNA. Further blind predictions of helix torsional properties, however, exposed inaccuracies in current BPLM theory, including three-fold discrepancies in torsional persistence length at the high force limit and the incorrect sign of dsRNA link-extension (twist-stretch) coupling. Beyond these experiments, HelixMC predicted that 'nucleosome-excluding' poly(A)/poly(T) is at least two-fold stiffer than random-sequence dsDNA in bending, stretching, and torsional behaviors; Z-DNA to be at least three-fold stiffer than random-sequence dsDNA, with a near-zero link-extension coupling; and non-negligible effects from base pair step correlations. We propose that experimentally testing these predictions should be powerful next steps for understanding the flexibility of dsDNA and dsRNA in sequence contexts and under mechanical stresses relevant to their biology.

Dataset Information

Correction-free force calibration for magnetic tweezers experiments.

Publications

Correction-free force calibration for magnetic tweezers experiments.

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