Single DNA/protein studies with magnetic traps
Introduction
In the past two decades, many different techniques to manipulate single molecules have been developed, such as atomic force microscopy and optical tweezers and magnetic traps. Magnetic traps consist of a set of magnets that provide a strong magnetic field gradient, thus exerting a force and a torque on magnetic beads tethered by a long biomolecule of interest (e.g. DNA, see Figure 1). The molecule extension is estimated by recording the 3D bead position with a conventional microscope with nanometer resolution [1]. These devices are now commercially available. The macroscopic magnets create a field gradient decaying exponentially over typically a fraction of a millimeter. Since the length of the molecule is in the micron range, force remains constant in a given experiment, whatever the changes in the molecule extension. Magnetic traps thus behave as force clamps, as they set the force applied on the bead, but not its position. By contrast, optical tweezers behave as position clamps: they impose the bead 3D position but not the force applied on it. Strong NdFeB magnets will pull a one-micron bead with forces up to 20 pN when placed at a hundred microns from that bead. Smaller forces are easily obtained upon moving the magnets away from the bead. Increasing the maximum force is achieved by using bigger (e.g. 2.8-microns) beads, yielding forces up to 160 pN. However, since the measurement noise is proportional to the viscous dissipation, smaller beads allow for better signal-to-noise ratio.
A very attractive feature of magnetic traps is their ability to apply a torque on the molecule of interest by rotating the magnets. Magnetic traps have emerged as a simple tool to study enzymes involving DNA supercoiling. Various magnet dispositions are used, providing for very different features. In the most common layout, the opposite poles of two magnets separated by a small gap generate a horizontal field with a strong vertical gradient. Rotating the magnets along the vertical axis keeps the pulling force constant while imposing a rotation angle and thus a magnetic torque. It turns out that this configuration displays a huge rotational stiffness (orders of magnitude larger than the DNA torsional stiffness), meaning that this type of magnetic traps behaves as a rotation clamp: the torsional angle on the molecule is fixed, while the torque can vary. This simple configuration allows one to impose DNA supercoiling but makes torque measurement difficult.
An alternative design uses a single magnetic pole (a sharp tip) where the field and its gradient are parallel and axi-symmetric [2•, 3, 4, 5]. In this configuration, a vertical force is still exerted, but the bead is free to rotate; torque can be measured by equilibrating it with the viscous drag on the rotating bead. Even though it is impossible to impose a rotation constraint in this situation, any deviation from the axi-symmetric configuration (inevitable in a real situation) will result in a small but non-zero torque on the bead, which might be useful in a variety of situations.
Various technical improvements have appeared recently, in particular addressing some drawbacks and coupling micromanipulation with single molecule fluorescence:
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In contrast to optical tweezers that act on a specific bead, magnetic traps are highly parallel: the magnets pull with the same force on all the beads located in the field of view; measurements are only limited by the number of beads whose position can be tracked simultaneously and their mutual magnetic interactions. Ribeck and Saleh have demonstrated the accurate tracking of 30 beads simultaneously [6]. Improvements in the measurement accuracy are under study [7].
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New anisotropic magnetic particles, exhibiting an easily detectable anisotropic image, associated with a magnet configuration with a nearly vertical field direction have allowed Celedon et al. [2•] to measure the torque associated with DNA and chromatin supercoiling. Alternatively, Zhang and Marko [8] have proposed to use the Maxwell relation in order to deduce the torque from extension-versus-rotation measurements in a rotation clamp configuration. The validity of this method was demonstrated experimentally [9].
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Force spectroscopy is ideal to probe molecular interactions: van Noort has proposed a simple method to measure the molecular internal interactions occurring at low forces (ca. 1 pN) [10].
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Fernandez and co-workers have pioneered the use of an electromagnet to stretch proteins [11]. Pulling on a single cytoplasmic protein exposes a binding site for other proteins, as revealed by TIRF, demonstrating the first steps of molecular mechanotransduction.
Micromanipulation experiments, and in particular magnetic traps experiments, can be used to investigate DNA/protein interactions if and only if this interaction affects the DNA extension, for example by stretching, bending, twisting or unwinding the molecule. In the following sections, we describe recent advances obtained in such studies using magnetic traps.
Section snippets
Their use in studying topologically modifying enzymes
Magnetic traps are particularly well adapted to investigate the activity of enzymes that control the topology of DNA molecules: type I and type II topoisomerases and gyrases. A magnetic bead tethered by one or two DNA molecules to a surface can be readily pulled and spun by the magnetic tweezers. The anchoring DNA molecule(s) can thus be easily and repeatedly coiled or braided with a subsequent decrease in the distance of the bead to the surface due to plectoneme formation. When a topoisomerase
Their use in studying DNA translocases
A DNA translocase is a motor using chemical energy to move along a DNA molecule. In general, such a translocation does not affect the DNA extension and thus cannot be studied with magnetic tweezers or any other micromanipulation technique unless the motor itself is bound to the surface, which was rarely done with magnetic traps [4, 5]. In the general case, only the initial binding and final unbinding of such a motor can be detected if it involves bending or twisting of the DNA molecule.
Their use in studying structural proteins, in particular chromatin
Magnetic tweezers have shown to be a powerful tool to investigate a wide range of structural proteins that associate with DNA and organize its structure. Several studies have given essential information concerning the way DNA is organized in vivo in prokaryotic as well as in eukaryotic cells. In Escherichia Coli, Skoko et al. have showed that the nucleoid protein Fis coats DNA to form an ordered array and stabilizes large DNA loops at or above 1 μM [46]. Lia et al. demonstrated that Heat
Others and conclusion
Other proteins interacting with DNA can be studied with magnetic tweezers: DNA polymerases, helicases [57, 58, 59], and so on. Particularly interesting examples are provided by the DNA repair enzymes with cruciform extrusion motors like RuvAB [3, 60, 61, 62], or recombination proteins like RecA [63, 64•, 65, 66] or Rad51 [67, 68]. It is to be expected that whenever torsion is playing a role in protein–DNA complexes, magnetic traps will provide a simple and useful means to study these
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We acknowledge the financial support of CNRS, ANR, IUF, HFSP and EU.
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