Combining optical tweezers and scanning probe microscopy to study DNA-protein interactions.
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.
Huisstede
Jurgen H G
JH
Biophysical Engineering Group and MESA Institute for Nanotechnology, Faculty of Science and Technology, University of Twente, AE Enschede 7500, The Netherlands.
Subramaniam
Vinod
V
Bennink
Martin L
ML
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism*; D004076:Digoxigenin / Q000378:metabolism*; D008846:Micromanipulation; D020527:Microscopy, Scanning Probe / Q000295:instrumentation* / Q000379:methods*; D052898:Optical Tweezers
D004279:DNA, Viral; D004076:Digoxigenin