Single molecule studies of helicases with magnetic tweezers

Hodeib, Samar; Raj, S. Shanmuga Sundara; Manosas, Maria; Zhang, Weiting; Bagchi, Debjani; Ducos, Bertrand; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

doi:10.1016/j.ymeth.2016.06.019

Cited by 25 publications

(13 citation statements)

References 46 publications

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“…Video-based three-dimensional (3D) tracking of microparticles has become a major tool revealing the dynamics of microorganism motility (1). On the other hand, by allowing to track the position of a microbead attached to a biopolymer, it is essential for the singlemolecule characterization of molecular motors such as polymerases or helicases (2). Compared to other force spectroscopic techniques such as optical tweezers or atomic force microscopes (AFM), techniques based on 3D tracking present the extremely useful advantage of being parallelizable.…”

Section: Introductionmentioning

confidence: 99%

Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

et al. 2021

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While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

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Section: Introductionmentioning

confidence: 99%

Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

et al. 2021

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“…Single-molecule techniques involving DNA or RNA, are powerful methods that provide detailed mechanistic insight into the structural transitions in nucleic acids under applied force as well as the interaction of a multitude of DNA or RNA binding proteins and their resultant effects on the nucleic acid being manipulated [1][2][3][4][5][6]. To achieve success, these techniques require high-quality, homogeneous preparations of the nucleic acid substrates ranging in size from 48504 bp (bacteriophage lambda DNA) to oligonucleotides 100 bp in length and shorter [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

High-yield purification of exceptional-quality, single-molecule DNA substrates

Bianco

2021

J Biol Methods

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Single-molecule studies involving DNA or RNA, require homogeneous preparations of nucleic acid substrates of exceptional quality. Over the past several years, a variety of methods have been published describing different purification methods but these are frustratingly inconsistent with variable yields even in the hands of experienced bench scientists. To address these issues, we present an optimized and straightforward, column-based approach that is reproducible and produces high yields of substrates or substrate components of exceptional quality. Central to the success of the method presented is the use of a non-porous anion exchange resin. In addition to the use of this resin, we encourage the optimization of each step in the construction of substrates. The fully optimized method produces high yields of a hairpin DNA substrate of exceptional quality. While this substrate is suitable for single-molecule, magnetic tweezer experiments, the described method is readily adaptable to the production of DNA substrates for the majority of single-molecule studies involving nucleic acids ranging in size from 70–15000 bp.

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“…[6][7][8] Important examples are optical tweezers (OT) and magnetic tweezers (MT) which have elucidated conformational changes of DNA 9 and mechanisms of DNA replication by helicases. 10 Optical tweezers have been successful in unravelling protein unfolding, 11,12 protein misfolding 13 and the kinetic properties of molecular motors such as kinesin 14 while further studies on the mechanics of cell surfaces are in progress. 15 In force spectroscopies, significant forces ( pN to nN) are applied to the molecule of interest, passed on by the handles that are attached to the molecule.…”

Section: Introductionmentioning

confidence: 99%