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

Abstract

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 similar to 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. (C) 2012 Elsevier Inc. All rights reserved