Skip to main content
SpringerLink
Log in

Unraveling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers

  • Protocol
  • First Online:
Bacterial Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1837))

Abstract

Genomes carry the genetic blueprint of all living organisms. Their organization requires strong condensation as well as carefully regulated accessibility to specific genes for proper functioning of their hosts. The study of the structure and dynamics of the proteins that organize the genome has benefited tremendously from the development of single-molecule force spectroscopy techniques that allow for real-time, nanometer accuracy measurements of the compaction of DNA and manipulation with pico-Newton scale forces. Magnetic tweezers in particular have the unique ability to complement such force spectroscopy with the control over the linking number of the DNA molecule, which plays an important role when DNA organizing proteins form or release wraps, loops, and bends in DNA. Here, we describe all the necessary steps to prepare DNA substrates for magnetic tweezers experiments, assemble flow cells, tether DNA to magnetics bead inside flow cell, and manipulate and record the extension of such DNA tethers. Furthermore, we explain how mechanical parameters of nucleo-protein filaments can be extracted from the data.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Luijsterburg MS, White MF, Van Driel R, Dame RT (2008) The major architects of chromatin: architectural proteins in bacteria, archaea and eukaryotes. Crit Rev Biochem Mol Biol 43:393–418

    Article  CAS  PubMed  Google Scholar 

  2. Bustamante C, Bryant Z, Smith SB (2003) Ten years of tension: single-molecule DNA mechanics. Nature 421:423–427

    Article  CAS  PubMed  Google Scholar 

  3. Smith S, Finzi L, Bustamante C (1992) Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science 258:1122–1126

    Article  CAS  PubMed  Google Scholar 

  4. Strick TR, Allemand JF, Bensimon D et al (1996) The elasticity of a single supercoiled DNA molecule. Science 271:1835–1837

    Article  CAS  PubMed  Google Scholar 

  5. Gosse C, Croquette V (2002) Magnetic tweezers: micromanipulation and force measurement at the molecular level. Biophys J 82:3314–3329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. van Noort J, Verbrugge S, Goosen N et al (2004) Dual architectural roles of HU: formation of flexible hinges and rigid filaments. Proc Natl Acad Sci U S A 101:6969–6974

    Article  PubMed  PubMed Central  Google Scholar 

  7. van Loenhout MTJ, van der Heijden T, Kanaar R et al (2009) Dynamics of RecA filaments on single-stranded DNA. Nucleic Acids Res 37:4089–4099

    Google Scholar 

  8. Lionnet T, Allemand JF, Revyakin A et al (2012) Single-molecule studies using magnetic traps. Cold Spring Harb Protoc 7:34–49

    Google Scholar 

  9. Lim CJ, Kenney LJ, Yan J (2014) Single-molecule studies on the mechanical interplay between DNA supercoiling and H-NS DNA architectural properties. Nucleic Acids Res 42:8369–8378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. van der Valk RA, Vreede J, Crémazy F, Dame RT (2014) Genomic looping: a key principle of chromatin organization. J Mol Microbiol Biotechnol 24:344–359

    Google Scholar 

  11. Meng H, Andresen K, Van Noort J (2015) Quantitative analysis of single-molecule force spectroscopy on folded chromatin fibers. Nucleic Acids Res 43:3578–3590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yan J, Skoko D, Marko JF (2004) Near-field-magnetic-tweezer manipulation of single DNA molecules. Phys Rev E Stat Nonlin Soft Matter Phys 70(1 Pt 1):011905

    Article  CAS  PubMed  Google Scholar 

  13. Berghuis BA, Kӧber M, van Laar T, Dekker NH (2016) High-throughput, high-force probing of DNA-protein interactions with magnetic tweezers. Methods 105:90–98

    Article  CAS  PubMed  Google Scholar 

  14. Ribeck N, Saleh OA (2008) Multiplexed single-molecule measurements with magnetic tweezers. Rev Sci Instrum 79(9):094301

    Article  CAS  PubMed  Google Scholar 

  15. De Vlaminck I, Henighan T, Van Loenhout MTJ et al (2011) Highly parallel magnetic tweezers by targeted DNA tethering. Nano Lett 11:5489–5493

    Article  CAS  PubMed  Google Scholar 

  16. 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 2:439

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lipfert J, Kerssemakers JWJ, Jager T, Dekker NH (2010) Magnetic torque tweezers: measuring torsional stiffness in DNA and RecA-DNA filaments. Nat Methods 7:977–980

    Article  CAS  PubMed  Google Scholar 

  18. Fisher JK, Cribb J, Desai KV et al (2006) Thin-foil magnetic force system for high-numerical-aperture microscopy. Rev Sci Instrum 77. https://doi.org/10.1063/1.2166509

  19. Oliver PM, Park JS, Vezenov D (2011) Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination. Nanoscale 3:581–591

    Article  CAS  PubMed  Google Scholar 

  20. Graham JS, Johnson RC, Marko JF (2011) Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res 39:2249–2259

    Article  CAS  PubMed  Google Scholar 

  21. Graham JS, Johnson RC, Marko JF (2011) Counting proteins bound to a single DNA molecule. Biochem Biophys Res Commun 415:131–134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Long X, Parks JW, Bagshaw CR, Stone MD (2013) Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy. Nucleic Acids Res 41:2746–2755

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Vilfan ID, Lipfert J, Koster DA et al (2009) Magnetic tweezers for single-molecule experiments. In: Hinterdorfer P, Oijen A (eds) Single-molecule biophysics. Springer, New York, pp 371–395

    Chapter  Google Scholar 

  24. te Velthuis AJW, Kerssemakers JWJ, Lipfert J, Dekker NH (2010) Quantitative guidelines for force calibration through spectral analysis of magnetic tweezers data. Biophys J 99:1292–1302

    Google Scholar 

  25. Yu Z, Dulin D, Cnossen J et al (2014) A force calibration standard for magnetic tweezers. Rev Sci Instrum 85(12):123114

    Article  CAS  PubMed  Google Scholar 

  26. Daldrop P, Brutzer H, Huhle A et al (2015) Extending the range for force calibration in magnetic tweezers. Biophys J 108:2550–2561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gupta P, Zlatanova J, Tomschik M (2009) Nucleosome assembly depends on the torsion in the DNA molecule: a magnetic tweezers study. Biophys J 97:3150–3157

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Vlijm R, Lee M, Lipfert J et al (2015) Nucleosome assembly dynamics involve spontaneous fluctuations in the handedness of tetrasomes. Cell Rep 10:216–225

    Article  CAS  PubMed  Google Scholar 

  29. Vlijm R, Lee M, Ordu O et al (2015) Comparing the assembly and handedness dynamics of (H3.3-H4)2 tetrasomes to canonical tetrasomes. PLoS One 10(10):e0141267

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kruithof M, Chien F, de Jager M, van Noort J (2008) Subpiconewton dynamic force spectroscopy using magnetic tweezers. Biophys J 94:2343–2348

    Article  CAS  PubMed  Google Scholar 

  31. Meng H, Bosman J, Van Der Heijden T, Van Noort J (2014) Coexistence of twisted, plectonemic, and melted DNA in small topological domains. Biophys J 106:1174–1181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Marko JF, Siggia ED (1995) Stretching DNA. Macromolecules 28:8759–8770

    Article  CAS  Google Scholar 

  33. Bustamante C, Smith SB, Liphardt J, Smith D (2000) Single-molecule studies of DNA mechanics. Curr Opin Struct Biol 10:279–285

    Article  CAS  PubMed  Google Scholar 

  34. Marko JF (2007) Torque and dynamics of linking number relaxation in stretched supercoiled DNA. Phys Rev E Stat Nonlin Soft Matter Phys 76(2 Pt 1):021926

    Article  CAS  PubMed  Google Scholar 

  35. Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271:795–798

    Article  CAS  PubMed  Google Scholar 

  36. Lowary P, Widom J (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J Mol Biol 276:19–42

    Article  CAS  PubMed  Google Scholar 

  37. Bancaud A, Wagner G, Conde e Silva N et al (2007) Nucleosome chiral transition under positive torsional stress in single chromatin fibers. Mol Cell 27:135–147

    Article  CAS  PubMed  Google Scholar 

  38. Chen CW, Thomas CA (1980) Recovery of DNA segments from agarose gels. Anal Biochem 101:339–341

    Article  CAS  PubMed  Google Scholar 

  39. Marko MA, Chipperfield R, Birnboim HC (1982) A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem 121:382–387

    Article  CAS  PubMed  Google Scholar 

  40. Boom R, Sol C, Salimans MMM et al (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28:495–503

    PubMed  PubMed Central  CAS  Google Scholar 

  41. Salvi G, De Los Rios P, Vendruscolo M (2005) Effective interactions between chaotropic agents and proteins. Proteins Struct Funct Genet 61:492–499

    Article  CAS  PubMed  Google Scholar 

  42. LePecq JB, Paoletti C (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol 27:87–106

    Article  CAS  PubMed  Google Scholar 

  43. Lee S-H, Roichman Y, Yi G-R et al (2007) Characterizing and tracking single colloidal particles with video holographic microscopy. Opt Express 15:18275

    Article  PubMed  Google Scholar 

  44. Gelles J, Schnapp BJ, Sheetz MP (1988) Tracking kinesin-driven movements with nanometre-scale precision. Nature 331:450–453

    Article  CAS  PubMed  Google Scholar 

  45. Lee GM, Ishihara A, Jacobson K (1991) Direct observation of brownian motion of lipids in a membrane. Proc Natl Acad Sci U S A 88:6274–6278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Anderson CM, Georgiou GN, Morrison IE et al (1992) Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C. J Cell Sci 101(Pt 2):415–425

    PubMed  Google Scholar 

  47. van Loenhout MTJ, Kerssemakers JWJ, De Vlaminck I, Dekker C (2012) Non-bias-limited tracking of spherical particles, enabling nanometer resolution at low magnification. Biophys J 102:2362–2371

    Google Scholar 

  48. Wong WP, Halvorsen K (2006) The effect of integration time on fluctuation measurements: calibrating an optical trap in the presence of motion blur. Opt Express 14:12517–12531

    Article  PubMed  Google Scholar 

  49. van der Horst A, Forde NR (2010) Power spectral analysis for optical trap stiffness calibration from high-speed camera position detection with limited bandwidth. Opt Express 18:7670–7677

    Article  CAS  PubMed  Google Scholar 

  50. Sitters G, Kamsma D, Thalhammer G et al (2014) Acoustic force spectroscopy. Nat Methods 12:47–50

    Article  CAS  PubMed  Google Scholar 

  51. Klaue D, Seidel R (2009) Torsional stiffness of single superparamagnetic microspheres in an external magnetic field. Phys Rev Lett 102:1–4

    Article  CAS  Google Scholar 

  52. Liu Y, Chen H, Kenney LJ, Yan J (2010) A divalent switch drives H-NS/DNA-binding conformations between stiffening and bridging modes. Genes Dev 24:339–344

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. van der Valk RA, Vreede J, Qin L et al (2017) Mechanism of environmentally driven conformational changes that modulate H-NS DNA bridging activity. elife 6:e27369

    Article  PubMed  PubMed Central  Google Scholar 

  54. Kulić IM, Mohrbach H, Thaokar R, Schiessel H (2007) Equation of state of looped DNA. Phys Rev E Stat Nonlin Soft Matter Phys 75(1 Pt 1):011913

    Article  CAS  PubMed  Google Scholar 

  55. Swinger KK, Rice PA (2004) IHF and HU: flexible architects of bent DNA. Curr Opin Struct Biol 14:28–35

    Article  CAS  PubMed  Google Scholar 

  56. Lin J, Chen H, Dröge P, Yan J (2012) Physical organization of DNA by multiple non-specific DNA-binding modes of integration host factor (IHF). PLoS One 7:e49885

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Luger K, Mäder AW, Richmond RK et al (1997) Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389:251–260

    Article  CAS  PubMed  Google Scholar 

  58. Schalch T, Duda S, Sargent DF, Richmond TJ (2005) X-ray structure of a tetranucleosome and its implications for the chromatin fibre. Nature 436:138–141

    Article  CAS  PubMed  Google Scholar 

  59. Song F, Chen P, Sun D et al (2014) Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units. Science 344:376–380

    Article  CAS  Google Scholar 

  60. Henneman B, Dame TR (2015) Archaeal histones: dynamic and versatile genome architects. AIMS Microbiol 1:72–81

    Article  Google Scholar 

  61. Mattiroli F, Bhattacharyya S, Dyer PN et al (2017) Structure of histone-based chromatin in Archaea. Science 357:609–612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Hatch K, Danilowicz C, Coljee V, Prentiss M (2008) Measurement of the salt-dependent stabilization of partially open DNA by Escherichia coli SSB protein. Nucleic Acids Res 36:294–299

    Article  CAS  PubMed  Google Scholar 

  63. Schäffet E, Nørrelykke SF, Howard J (2007) Surface forces and drag coefficients of microspheres near a plane surface measured with optical tweezers. Langmuir 23:3654–3665

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Huygens-Kamerlingh Onnes Laboratory, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, Leiden, CA, The Netherlands

    Thomas B. Brouwer, Artur Kaczmarczyk, Chi Pham & John van Noort

Authors
  1. Thomas B. Brouwer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Artur Kaczmarczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chi Pham
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John van Noort
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John van Noort .

Editor information

Editors and Affiliations

  1. Leiden Institute of Chemistry and Centre for Microbial Cell Biology, Leiden University, Leiden, The Netherlands

    Remus T. Dame

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Brouwer, T.B., Kaczmarczyk, A., Pham, C., van Noort, J. (2018). Unraveling DNA Organization with Single-Molecule Force Spectroscopy Using Magnetic Tweezers. In: Dame, R. (eds) Bacterial Chromatin. Methods in Molecular Biology, vol 1837. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8675-0_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8675-0_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8674-3

  • Online ISBN: 978-1-4939-8675-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation