title: 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 ∼20pN before being pulled free from the bilayer, whereas molecules anchored to the bilayer through multiple attachment points can withstand ⩾65pN; 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.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, NY 10032, USA .
authors.forename: Ja Yil
authors.initials: JY
authors.lastname: Lee
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Teresa
authors.initials: T
authors.lastname: Fazio
authors.forename: Shalom
authors.initials: S
authors.lastname: Wind
authors.forename: Eric C
authors.initials: EC
authors.lastname: Greene
mesh_terms: D004247:DNA / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D052898:Optical Tweezers; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D011506:Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases