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Constructing a Magnetic Tweezers to Monitor RNA Translocation at the Single-Molecule Level

  • Desiree Salas
  • Veronika Gocheva
  • Marcelo NöllmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

Single-molecule methods have become an invaluable tool in the investigation of the mechanisms of nucleic-acid motors. Magnetic tweezers is a single-molecule manipulation technique that permits the real-time measurement of enzyme activities on single nucleic-acid molecules at high-resolution, high-throughput, and inherently constant force. Here, we describe several aspects of the implementation of magnetic tweezers, with special emphasis on the construction of a simple magnetic trap and, in particular, on the detailed description of image analysis methods to measure the extension changes in nucleic-acid molecules induced by protein activity. Finally, we carefully describe the steps involved in performing a full magnetic tweezers experiment.

Key words

Motors Nucleic-acid enzymes RNA Magnetic tweezers Single molecule Mechanochemistry 

Notes

Acknowledgments

We thank Francesco Pedaci and Antoine Le Gall for critical reading and very helpful comments. Financial support was provided by the Human Frontiers Science Program (M.N.) and the European Research Council (Starting Grant 260787 to M.N.).

References

  1. 1.
    te Velthuis AJW, Kerssemakers JWJ, Lipfert J et al (2010) Quantitative Guidelines for Force Calibration through Spectral Analysis of Magnetic Tweezers Data. Biophys J 99:1292–1302CrossRefGoogle Scholar
  2. 2.
    Lionnet T, Allemand JF, Revyakin A et al (2012) Single-molecule studies using magnetic traps. Cold Spring Harb Protoc 2012:34–49PubMedGoogle Scholar
  3. 3.
    Vilfan ID, Lipfert J, Koster DA et al (2009) Magnetic Tweezers for Single-Molecule Experiments, Handbook of Single-Molecule Biophysics. Springer, New YorkGoogle Scholar
  4. 4.
    Lansdorp BM, Saleh OA (2012) Power spectrum and Allan variance methods for calibrating single-molecule video-tracking instruments. Rev Sci Instrum 83:025115PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Strick T, Allemand J, Croquette V et al (2000) Twisting and stretching single DNA molecules. Prog Biophys Mol Biol 74:115–140PubMedCrossRefGoogle Scholar
  6. 6.
    Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271:795–799PubMedCrossRefGoogle Scholar
  7. 7.
    Bustamante C, Marko JF, Siggia ED et al (1994) Entropic elasticity of lambda-phage DNA. Science 265:1599–1600PubMedCrossRefGoogle Scholar
  8. 8.
    Strick TR, Allemand JF, Bensimon D et al (1996) The elasticity of a single supercoiled DNA molecule. Science 271:1835–1837PubMedCrossRefGoogle Scholar
  9. 9.
    Herrero-Galán E, Fuentes-Perez ME, Carrasco C et al (2013) Mechanical identities of RNA and DNA double helices unveiled at the single-molecule level. J Am Chem Soc 135:122–131PubMedCrossRefGoogle Scholar
  10. 10.
    Abels JA, Moreno-Herrero F, van der Heijden T et al (2005) Single-molecule measurements of the persistence length of double-stranded RNA. Biophys J 88:2737–2744PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Liphardt J, Onoa B, Smith SB et al (2001) Reversible unfolding of single RNA molecules by mechanical force. Science 292:733–737PubMedCrossRefGoogle Scholar
  12. 12.
    Lipfert J, Wiggin M, Kerssemakers JWJ et al (2011) Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids. Nat Commun 2:439PubMedCrossRefGoogle Scholar
  13. 13.
    Lebel P, Basu A, Oberstrass FC et al (2014) Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension. Nat Methods 11:456–462PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Bryant Z, Oberstrass FC, Basu A (2012) Recent developments in single-molecule DNA mechanics. Curr Opin Struct Biol 22:304–312PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Mosconi F, Allemand JF, Bensimon D et al (2009) Measurement of the torque on a single stretched and twisted DNA using magnetic tweezers. Phys Rev Lett 102:078301PubMedCrossRefGoogle Scholar
  16. 16.
    Crisona NJ, Strick TR, Bensimon D et al (2000) Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements. Genes Dev 14:2881–2892PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Stone MD, Bryant Z, Crisona NJ et al (2003) Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases. Proc Natl Acad Sci U S A 100:8654–8659PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Koster DA, Croquette V, Dekker C et al (2005) Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 434:671–674PubMedCrossRefGoogle Scholar
  19. 19.
    Nollmann M, Stone MD, Bryant Z et al (2007) Multiple modes of Escherichia coli DNA gyrase activity revealed by force and torque. Nat Struct Mol Biol 14:264–271PubMedCrossRefGoogle Scholar
  20. 20.
    Ribeck N, Saleh OA (2008) Multiplexed single-molecule measurements with magnetic tweezers. Rev Sci Instrum 79:094301PubMedCrossRefGoogle Scholar
  21. 21.
    De Vlaminck I, Henighan T, van Loenhout MTJ et al (2011) Highly Parallel Magnetic Tweezers by Targeted DNA Tethering. Nano Lett 11:5489–5493PubMedCrossRefGoogle Scholar
  22. 22.
    Ding F, Manosas M, Spiering MM et al (2012) Single-molecule mechanical identification and sequencing. Nat Methods 9:367–372PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Manosas M, Perumal SK, Croquette V et al (2012) Direct observation of stalled fork restart via fork regression in the T4 replication system. Science 338:1217–1220PubMedCrossRefGoogle Scholar
  24. 24.
    Manosas M, Meglio A, Spiering MM et al (2010) Magnetic Tweezers for the Study of DNA Tracking Motors. Methods Enzymol 475:297–320PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Lionnet T, Allemand JF, Revyakin A et al. (2011) Magnetic trap construction. Cold Spring Harb Protoc 2012, pdb.prot067496–pdb.prot067496Google Scholar
  26. 26.
    Revyakin A, Ebright RH, Strick TR (2005) Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments. Nat Methods 2:127–138PubMedCrossRefGoogle Scholar
  27. 27.
    Cheng W, Dumont S, Tinoco I et al (2007) NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork. Proc Natl Acad Sci U S A 104:13954–13959PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Ovryn B, Izen S (2000) Imaging of transparent spheres through a planar interface using a high-numerical-aperture optical microscope. J Opt Soc Am A Opt Image Sci Vis 17:1202–1213PubMedCrossRefGoogle Scholar
  29. 29.
    Gosse C, Croquette V (2002) Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys J 82:3314–3329PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    van Loenhout MTJ, Kerssemakers JWJ, De Vlaminck I et al (2012) Non-Bias-Limited Tracking of Spherical Particles, Enabling NanometerResolution at Low Magnification. Biophys J 102:2362–2371PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Lipfert J, Kerssemakers JJW, Rojer M et al (2011) A method to track rotational motion for use in single-molecule biophysics. Rev Sci Instrum 82:103707PubMedCrossRefGoogle Scholar
  32. 32.
    Cattoni DI, Fiche J-B, Valeri A et al (2013) Super-resolution imaging of bacteria in a microfluidics device. PLoS One 8:e76268PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    De Vlaminck I, Henighan T, van Loenhout MTJ et al (2012) Magnetic Forces and DNA Mechanics in Multiplexed Magnetic Tweezers. PLoS One 7:e41432PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Ptacin JL, Nollmann M, Becker EC et al (2008) Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis. Nat Struct Mol Biol 15:485–493PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Desiree Salas
    • 1
    • 2
    • 3
  • Veronika Gocheva
    • 1
    • 2
    • 3
  • Marcelo Nöllmann
    • 1
    • 2
    • 3
    Email author
  1. 1.Centre de Biochimie StructuraleCNRS UMR5048MontpellierFrance
  2. 2.INSERM U1054MontpellierFrance
  3. 3.Universités Montpellier I et IIMontpellierFrance

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