Huisstede Jurgen H G, Subramaniam Vinod, Bennink Martin L
Biophysical Engineering Group and MESA Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, AE Enschede 7500, The Netherlands.
Microsc Res Tech. 2007 Jan;70(1):26-33. doi: 10.1002/jemt.20382.
We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm.
我们展示了使用一种新设计和制造的仪器在单分子水平研究DNA-蛋白质相互作用所获得的首批结果。这种显微镜将光镊与扫描探针显微镜相结合，使我们能够在单个悬浮的DNA分子上定位DNA结合蛋白。使用光镊将单个DNA分子拉紧，同时沿分子扫描一个探针。用光镊测量探针与样品之间的相互作用力。因此，该仪器使我们能够将结合蛋白的机械和功能特性与DNA分子内的张力联系起来。发现用作探针的微量移液器与裸露的DNA分子之间的典型摩擦力小于1皮牛。一个16微米的DNA分子，在DNA中间90纳米范围内大约有10-15个地高辛（DIG）分子，被用作模型系统。通过用抗地高辛（α-DIG）抗体包被的移液器进行扫描，我们能够利用该抗体-抗原对之间的高结合亲和力来定位这些位点。假设探针无限薄且刚性以及单个α-DIG/DIG键，估计的实验分辨率为15纳米。
