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Magnetic Torque Tweezers for Single Molecule Measurements of DNA-Ligand...

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Titel Magnetic Torque Tweezers for Single Molecule Measurements of DNA-Ligand and DNA-Protein Interactions
Looptijd 02 / 2010 - 02 / 2013
Status Lopend
Onderzoeknummer OND1337446
Leverancier gegevens NWO

Samenvatting

Koppel - het samenstel van krachten die ontstaan door rotatie - speelt een belangrijke rol in de biologie. Zo leidt de replicatie van DNA als gevolg van de helixstructuur tot opbouw van torsie in DNA. Het doel van dit project is om een magnetische pincet te ontwikkelen waarmee de draaimomenten van enkele DNA-moleculen gemeten kunnen worden. Hiermee kan de interactie van DNA-moleculen met eiwitten en medicijnen bestudeerd worden.

Samenvatting (EN)

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.

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Projectleider Dr. J. Lipfert

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