کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1929280 | 1050451 | 2012 | 6 صفحه PDF | دانلود رایگان |
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.
► A hybrid system to combine DNA curtains and optical trap is developed.
► The rupture force of a single lipid out of lipid bilayer is estimated.
► Multiple biotin/dig handles enables DNA to be extend to B–S transition region.
► Promoter-bound RNAP is disrupted by stretching DNA at physiological salt condition.
Journal: Biochemical and Biophysical Research Communications - Volume 426, Issue 4, 5 October 2012, Pages 565–570