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Measuring Two at the Same Time: Combining Magnetic Tweezers with Single-Molecule FRET

  • Marko Swoboda
  • Maj Svea Grieb
  • Steffen Hahn
  • Michael SchlierfEmail author
Chapter
Part of the Experientia Supplementum book series (EXS, volume 105)

Abstract

Molecular machines are the workhorses of the cell that efficiently convert chemical energy into mechanical motion through conformational changes. They can be considered powerful machines, exerting forces and torque on the molecular level of several piconewtons and piconewton-nanometer, respectively. For studying translocation and conformational changes of these machines, fluorescence methods, like FRET, as well as “mechanical” methods, like optical and magnetic tweezers, have proven well suited over the past decades. One of the current challenges in the field of molecular machines is gaining maximal information from single-molecule experiments by simultaneously measuring translocation, conformational changes, and forces exerted by these machines. In this chapter, we describe the combination of magnetic tweezers with single-molecule FRET for orthogonal simultaneous readout to maximize the information gained in single-molecule experiments.

Keywords

Magnetic tweezers and single-molecule FRET DNA manipulation Conformational changes Molecular machines 

Abbreviations

CCD

Charge-coupled device

DM

Dichroic mirror

DNA

Deoxyribonucleic acid

dNTP

Deoxyribonucleoside triphosphate

dTTP

Deoxythymidine triphosphate

dUTP

Deoxyuridine triphosphate

EMCCD

Electron multiplying charge-coupled device

FRET

Förster resonance energy transfer

GPU

Graphics processing unit

HJ

Holliday junction

LUT

Lookup table

PCR

Polymerase chain reaction

RCLED

Resonant-cavity light-emitting diode

RNA

Ribonucleic acid

ROI

Region of interest

TIRF

Total internal reflection fluorescence

Notes

Acknowledgements

We gratefully acknowledge numerous discussions with the group of Ralf Seidel, in particular Ralf Seidel, Alexander Huhle, and Friedrich Schwarz. Furthermore, discussions and software support by members of the Schlierf lab are highly appreciated. This work was supported by grants from BMBF 03Z2EN11 (to M.S.).

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Copyright information

© Springer Basel 2014

Authors and Affiliations

  • Marko Swoboda
    • 1
  • Maj Svea Grieb
    • 1
  • Steffen Hahn
    • 1
  • Michael Schlierf
    • 1
    Email author
  1. 1.BCUBE - Center for Molecular BioengineeringTechnische Universität DresdenDresdenGermany

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