Wednesday, September 12, 2012

Measuring intermolecular rupture forces with a combined TIRF-optical trap microscope and DNA curtains

Ja Yil Lee, Feng Wang, Teresa Fazio, Shalom Wind, Eric Greene

We report a new approach to probing DNA–protein interactions by combining optical tweezers with a high-throughput DNA curtains technique. Here we determine the forces required to remove the individual lipid-anchored DNA molecules from the bilayer. We demonstrate that DNA anchored to the bilayer through a single biotin–streptavidin linkage withstands ∼20 pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65 pN; access to this higher force regime is sufficient to probe the responses of protein–DNA interactions to force changes. As a proof-of-principle, we concurrently visualized DNA-bound fluorescently-tagged RNA polymerase while simultaneously stretching the DNA molecules. This work presents a step towards a powerful experimental platform that will enable concurrent visualization of DNA curtains while applying defined forces through optical tweezers.

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