A high-resolution magnetic tweezer for single-molecule measurements

Materials Department, University of California, Santa Barbara, CA 93106, USA.
Nucleic Acids Research (Impact Factor: 9.11). 10/2009; 37(20):e136. DOI: 10.1093/nar/gkp725
Source: PubMed


Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered
magnetic bead while using optical bead tracking to measure the biomolecule’s extension. While relatively simple to set up,
prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes.
Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than
traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces
that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position
to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.

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    • "When conducting single-molecule mechanical studies, there are a variety of techniques to apply and measure force [1]. Methods such as optical and magnetic tweezers typically require binding of a molecule to a surface (e.g. a glass slide, bead, etc.) [2] [3] [4] [5]. Irrespective of the type of attachment, the lifetime of the attachment bond is strongly dependent on the applied force [6]. "
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    ABSTRACT: Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA-protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (> 60pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.
    Preview · Article · Mar 2011 · Colloids and surfaces B: Biointerfaces
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    • "The ligation efficiency strongly depends on the length of the sticky end of the handle and it is rather low for the typical 4 nt overhangs generated by restriction enzymes. Longer overhangs can be generated using Autosticky PCR (37), which supports efficient ssDNA to dsDNA (38). However, this method is restricted to 5′-overhangs only and requires rather expensive primers. "
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    ABSTRACT: Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required.
    Full-text · Article · Nov 2010 · Nucleic Acids Research
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    • "In the study of DNA associated proteins and enzymes the Magnetic Tweezer technique has been found to be a powerful tool [32], it comprises a single DNA molecule anchored at one end to a surface and, at the other end to a micron-scale magnetic bead [33 – 38]. Magnets are used to pull and rotate the microbead, thus stretching and twisting the DNA molecule. "
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    ABSTRACT: This work was initiated by single molecule studies of the molecular motor activity of the Type I Restriction – Modification enzyme EcoR124I at a time when the consensus viewpoint was that such motors could not manipulate microscale objects. Type I Restriction-Modification enzymes process DNA, prior to cleavage, by means of translocation and this work was described using such single molecule studies involving the use of a Magnetic Tweezer setup (which also demonstrated that a micron-sized magnetic bead could be pulled over several microns distance by the 20 nm motor). Recently, this initial work has led to the development of an electronic version of the Magnetic Tweezer setup, which can also manipulate the DNA-attached bead, allowing its use as a biosensor and tool for drug discovery. The system can be used for a wide range of DNA-manip-ulating enzymes, many of which are potential drug targets.
    Full-text · Conference Paper · May 2010
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