Advertisement

Transverse Magnetic Tweezers Allowing Coincident Epifluorescence Microscopy on Horizontally Extended DNA

  • Stephen J. Cross
  • Claire E. Brown
  • Christoph G. BaumannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1431)

Abstract

Longitudinal magnetic tweezers (L-MT) have seen wide-scale adoption as the tool-of-choice for stretching and twisting a single DNA molecule. They are also used to probe topological changes in DNA as a result of protein binding and enzymatic activity. However, in the longitudinal configuration, the DNA molecule is extended perpendicular to the imaging plane. As a result, it is only possible to infer biological activity from the motion of the tethered superparamagnetic microsphere. Described here is a “transverse” magnetic tweezers (T-MT) geometry featuring simultaneous control of DNA extension and spatially coincident video-rate epifluorescence imaging. Unlike in L-MT, DNA tethers in T-MT are extended parallel to the imaging plane between two micron-sized spheres, and importantly protein targets on the DNA can be localized using fluorescent nanoparticles. The T-MT can manipulate a long DNA construct at molecular extensions approaching the contour length defined by B-DNA helical geometry, and the measured entropic elasticity agrees with the worm-like chain model (force < 35 pN). By incorporating a torsionally constrained DNA tether, the T-MT would allow both the relative extension and twist of the tether to be manipulated, while viewing far-red emitting fluorophore-labeled targets. This T-MT design has the potential to enable the study of DNA binding and remodeling processes under conditions of constant force and defined torsional stress.

Key words

Transverse magnetic tweezers Coincident fluorescence microscopy DNA micromanipulation Single-molecule manipulation 

Notes

Acknowledgements

The authors would like to thank the University of York Biology Electronic and Mechanical Workshops, especially M. Bentley for custom fabrications and S.P. Howarth for stepper motor control software. CGB would like to thank H.K.H. Fung, D. Jones, D.J. Richardson, and J.F. Watson for comments on the manuscript and assistance with method development. SJC and CEB were supported by a BBSRC PhD studentship and Genetics Society Summer Studentship, respectively. T-MT construction and development was supported by the BBSRC and the Department of Biology, University of York.

References

  1. 1.
    De Vlaminck I, Dekker C (2012) Recent advances in magnetic tweezers. Annu Rev Biophys 41:453–472CrossRefPubMedGoogle Scholar
  2. 2.
    Strick TR, Allemand J-F, Bensimon D, Bensimon A, Croquette V (1996) The elasticity of a single supercoiled DNA molecule. Science 271:1835–1837CrossRefPubMedGoogle Scholar
  3. 3.
    Bryant Z, Oberstrass FC, Basu A (2012) Recent developments in single-molecule DNA mechanics. Curr Opin Struct Biol 22:304–312CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Danilowicz C, Coljee VW, Bouzigues C, Lubensky DK, Nelson DR, Prentiss M (2003) DNA unzipping under a constant force exhibits multiple metastable intermediates. Proc Natl Acad Sci U S A 100:1694–1699CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Graham JS, Johnson RC, Marko JF (2011) Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA. Nucleic Acids Res 39:2249–2259CrossRefPubMedGoogle Scholar
  6. 6.
    Schwarz FW, Toth J, van Aelst K, Cui G, Clausing S, Szczelkun MD et al (2013) The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA. Science 340:353–356CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    van Loenhout MTJ, de Grunt MV, Dekker C (2012) Dynamics of DNA supercoils. Science 338:94–97CrossRefPubMedGoogle Scholar
  8. 8.
    Haber C, Wirtz D (2000) Magnetic tweezers for DNA micromanipulation. Rev Sci Instrum 71:4561–4570CrossRefGoogle Scholar
  9. 9.
    Neuman KC, Nagy A (2008) Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods 5:491–505CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chiou C-H, Huang Y-Y, Chiang M-H, Lee H-H, Lee G-B (2006) New magnetic tweezers for investigation of the mechanical properties of single DNA molecules. Nanotechnology 17:1217–1224CrossRefGoogle Scholar
  11. 11.
    Baumann CG, Bloomfield VA, Smith SB, Bustamante C, Wang MD, Block SM (2000) Stretching of single collapsed DNA molecules. Biophys J 78:1965–1978CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Strick TR, Allemand J-F, Bensimon D, Croquette V (1998) Behavior of supercoiled DNA. Biophys J 74:2016–2028CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Bustamante C, Marko JF, Siggia ED, Smith SB (1994) Entropic elasticity of lambda-phage DNA. Science 265:1599–1600CrossRefPubMedGoogle Scholar
  14. 14.
    Baumann CG, Smith SB, Bloomfield VA, Bustamante C (1997) Ionic effects on the elasticity of single DNA molecules. Proc Natl Acad Sci U S A 94:6185–6190CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Seol Y, Neuman KC (2011) Single-molecule measurements of topoisomerase activity with magnetic tweezers. Methods Mol Biol 778:229–241CrossRefPubMedGoogle Scholar
  16. 16.
    Baumann CG, Cross SJ (2011) Probing the mechanics of the complete DNA transcription cycle in real-time using optical tweezers. Methods Mol Biol 778:175–191CrossRefPubMedGoogle Scholar
  17. 17.
    Cross SJ (2013) Combining magnetic tweezers and single-molecule fluorescence microscopy to probe transcription-coupled DNA supercoiling. PhD thesis, University of York, YorkGoogle Scholar
  18. 18.
    Oliver PM, Park JS, Vezenov D (2011) Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination. Nanoscale 3:581–591CrossRefPubMedGoogle Scholar
  19. 19.
    Cheezum MK, Walker WF, Guilford WH (2001) Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys J 81:2378–2388CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Stephen J. Cross
    • 1
  • Claire E. Brown
    • 1
  • Christoph G. Baumann
    • 1
    Email author
  1. 1.Department of BiologyUniversity of YorkHeslington, YorkUK

Personalised recommendations