Magnetic Torque Tweezers for Single Molecule Measurements of DNA-Ligand and DNA-Protein Interactions

Samenvatting

Magnetic tweezers are a single molecule technique that permits to apply forces and torques to biological macromolecules. While the forces applied in magnetic tweezers set-ups can be quantitatively calibrated by measuring the Brownian fluctuations of trapped molecules, direct measurement of the applied torques is currently impossible. Here, I propose to develop magnetic torque tweezers that will permit to directly measure the applied torque and enable a new class of ?torque spectroscopy? measurements.

The torque measurement relies on tracking of the rotation angle of tethered beads by video microscopy and on the design of a novel magnet geometry. The capability to directly measure torque will leverage the advantages of existing magnetic tweezers, namely straightforward control of the supercoiled state of DNA for both negative and positive supercoils, facile extension to parallel measurements of multiple molecules, and a simple and robust experimental set-up.
Magnetic torque tweezers will provide a unique tool to address problems of biological interest and I propose to apply the method to DNA-ligand interactions, to the relaxation of DNA supercoils by type I topoisomerases, and to a range of other potential targets. These measurements will elucidate the role of torque in small molecule binding to DNA and will provide unique insights into the action of type I topoisomerases, in particular in the presence of topoisomerase inhibitors, which are currently used clinically as anti-tumor drugs. Ultimately, the proposed magnetic torque tweezers have the potential to more generally illuminate the role of torque in cellular processes such as transcription and replication.

Output

Wetenschappelijk artikel

  • N.H. Dekker, T. Jager, J.W.J. Kerssemakers, J. Lipfert(2010): Magnetic Torque Tweezers: Measuring Torsional Stiffness of DNA and RecA-DNA filaments Nature Methods pp. 977 - 980
  • S. Klijnhout, J. Lipfert, N.H. Dekker(2010): Torsional Sensing of Small-Molecule Binding Using Magnetic Tweezers Nucleic Acids Research pp. 7122 - 7132
  • J.W.J. Kerssemakers, J. Lipfert, A. J. W. te Velthuis, N.H. Dekker(2010): Quantitative guidelines for force calibration through spectral analysis of magnetic tweezers data Biophysical Journal pp. 1292 - 12302
  • J. Lipfert, J.W.J. Kerssemakers, M. Rojer, N.H. Dekker(2011): A method to track rotational motion for use in single-molecule biophysics Review of Scientific Instruments pp. 103707 - 103717
  • J.W.J. Lipfert, M. Wiggin, J.W.J. Kerssemakers, F. Pedaci, N.H. Dekker(2011): Freely-Orbiting Magnetic Tweezers to Directly Monitor Changes in the Twist of Nucleic Acids Nature Communications pp. 439 - 447
  • AY Sim, J Lipfert, D Herschlag, S Doniach(2012): Salt dependence of the radius of gyration and flexibility of single-stranded DNA in solution probed by small-angle x-ray scattering Physical Review E pp. 021901 - 021905
  • XJ Janssen, J Lipfert, T Jager, R Daudey, J Beekman, NH Dekker(2012): Electromagnetic torque tweezers: a versatile approach for measurement of single-molecule twist and torque Nano Letters pp. 3634 - 3639
  • S Doniach, J Lipfert(2012): Small and Wide Angle X-ray Scattering from Biological Macromolecules and their Complexes in Solution Comprehensive Biophysics pp. 376 - 397
  • D Dulin, J Lipfert, MC Moolman, NH Dekker(2012): Studying genomic processes at the single-molecule level: introducing the tools and applications Nature Reviews Genetics pp. 9 - 22
  • R.C. Oliver, J. Lipfert, D.A. Fox, R.H. Lo, S. Doniach, L. Columbus(2013): Dependence of micelle size and shape on detergent alkyl chain length and head group PLOS ONE pp. e62488 - e62488
  • M. Lee, J. Lipfert, H Sanchez, C Wyman, N.H. Dekker(2013): Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament Nucleic Acids Research pp. 7023 - 7230

Professionele publicatie

  • C. Menze, J. Lipfert, G. Harsch, B. Menze, S. Menze(2010): Der Vorfahr aus der Bronzezeit

Publicatie bedoeld voor een breed publiek

Kenmerken

Projectnummer

680-47-408

Hoofdaanvrager

Dr. J. Lipfert

Verbonden aan

Ludwig-Maximilians-Universit√§t M√ľnchen, Abteilung Angewandte Physik

Uitvoerders

Dr. J. Lipfert

Looptijd

01/02/2010 tot 24/04/2014